Sử dụng vector pHT01 để khảo sát sự biểu hiện tiết protein chỉ thị α amylase trong bacillus subtilis WB800N

Tgp chi Cong nghe Sinh hoc 11(2): 327-332, 2013

Se DUNG VECTOR pHT01 DE KHAO SAT STY BIEU HIEN TIET PROTEIN CHI THI
a-AMYLASE TRONG BACILLUS SUBTILIS WB800N
Phan Thi Phtrqng Trang, Nguyen HoM Nam, Trill Linh Think, Nguyen Dtic Huang
Triton Dgi hoc Khoa hoc ty. nhien, Dai hoc Qujc gia Thanh phi!) 1-1j Chi Minh
TOM TAT
Su xuat hien plasmid cam Ong thin:mg mai dau tien, pHT01 ben ve mat cau trite da thitc day khuynh huong
nghien dm cam ling bleu hien war tnirc protein tai to hgp cho Bacillus subtilis (B. subtilis). Cac nghien dm co
lien quan den plasmid nay deu &we thuc hien tren cac Chung B. subtilis co nguon goc a 168, nhu B. subtilis
1012, nhung chua dugc thuc hiOn trong Chung B. subtilis WB800N — chimg bi dot bien bat hoat 8 protease
ngoai bao. De than' de, kha rang s6 dung plasmid pHT01 vai B. subtilis WB800N trong bieu hien protein tiet
vugt mirc, chimg fed sir dung chi thi a-amylase tiet dugc ma hem bgi amyQ dui Bacillus amyloliquefasciens.
Plasmid pHT43-amyQ dugc tao ra va sir dkuig de khao sat bieu hien tiet a-amylase trong hai chimg B. subtilis
1012 va B. subtilis WB800N. Ket qua cho thay kha nang bieu hien a-amylase tiet hien qua trong ca hai chimg
B. subtilis, trong de lugng protein tao ra 6 WB800N co phin cao han 1012. Dieu nay cho thay ti'em rang sir
dung plasmid pHT01 vgi B. subtilis WB800N cho muc dich bleu hien protein tai to hgp tiet.
Trr khda: amyQ, Bacillus subtilis, plasmid pHT, pHT01, promoter Pgrac, WB800N.

M6 DAU
Chi& lugc san xuat protein tai .t6 hop tir cac
cluing vi sinh vat dang dugc phat. trien mph va da
dat dugc nhtrng thanh tru ding ke trong nhieu nam
qua. Co the nil, Escherichia coli (E. coli) dugc sir
dung nhtr mot nha may san xuat protein tai to hgp tir
rat sam va khfi pho bien cho den ngay nay (Peti,
Page, 2007). Nhung ben canh nhung uu diem nhu
th8ng tin di truyen dugc hieu rO, thao tic don gian,
co san nhieu loai vector ding nhu cac ching dOt bien
co lqi thi E. coli lai btic to mot so bat lgi do tich buy
dOc to, gay benh a ngoai va khong co kha nang tiet

protein hieu qua (Petsch, Anspach, 2000). Vi Vay cac
chung vi sinh vat an toan hors dang dugc chi tong
phit trien, tieu bieu la Bacillus subtilis (B. subtilis),
mot sinh vat mo hinh cia vi khufin Gram duong. Vi
khufin nay co nhieu tru diem nhu: (i) ching vi sinh
vat an toan dung trong thuc pham, dugc to chirc
FDA (Food and Drug Administration) eau my dfinh
gia la thuc nhOm vi sinh vat an toan; (ii) co kha
nang len men a mat d6 cao; (iii) co kha nang tiet
hieu qua, protein ra ngoai to bao, de dang cho viec
tinh che protein muc tieu (Harwood, 1992;
Schallmey et al., 2004; Schumann, 2007). Hien nay,
s6 lugng cac ching B., subtilis dugc irng dung trong
nghien ciru va san xuat ngay cang tang. Bang cich
tao ra nhiing dOt bien có lgi, cac nha khoa hoc da tao
nhu cau
ra nhieu chimg B. subtilis mai phi hop
sir dung. Tieu bieu trong so do la cac chin mang

di5t Nen tren cac protease ngoai bao nhu B. subtilis
WB800 (Wu et al., 2002). Nhung vi WB800 mang
gene khfing chloramphenicol (Cm), khong tuang
(Nguyen
.
thich vii ding plasmid pHT, nen. WB800N
khac
phuc
nhugc
et .al., 2011) da dugc tao ra
diem nay.

a

Promoter du nen dugc sit dung kiem sok su
bieu hien tren B. subtilis là Pspac, cam irng bang
Isopropyl (3-D- I -thiogalactopyranoside (IPTG)
(Yansura, Henner, 1984)., Tiep sau Q6 la mot boat cac
he th8ng sir dung cac chat cam irng nhu tetracycline
(GeissendOrfer, Hillen, 1990), xylose (Kim et al.,
1996), citrate (Yamamoto et al., 2000), subtilin
(Bongers et al., 2005), glycine (Phan, Schumann,
2007) va he thong khfic dua teen T7 RNA
polymerase (Chen et al., 2010). Gan day, ching toi
tao ra promoter cam irng mai cho B. subtilis throe
dat ten la Pgrac (Phan et al., 2006), dung hop tir
promoter with groESL co ngtion gee tir B. subtilis
vii lac operator (lacO) tir E. coli, sir dung chat cam
img la IPTG, cho kha nang bieu hien protein muc
tieu len den 16% protein tong so, gap 50 ban so vii
Pspac (Phan et al., 2012). He thong plasmid dau tien
dua teen promoter nay la pHT01 (Nguyen et al.,
2007), mot trong nhung plasmid dugc‘phan plied bai
Mobitec. Tuy nhien, cac th6ng tin ye khi nang sir
dung cac plasmid nay co nguOn goc tir promoter
Pgrac nay vii cac ching vi khufin khac nhau van con
rat han che.
327


Phan Thi Ph uvng Trang et al.

Trong nghien cull nay, nhim mac dich khao sat
kha nang sir dung plasmid pHT01 de bleu hien
protein tiet trong chimg chti mai B. subtilis
WB800N, chimg t6i sir dung gene chi thi amyQ ma
Ilea enzyme a-amylase tir B. amyloliquefasciens
(Palva, 1982). Day la met chi thi dugc sir dung kha
phibien trong cac nghien cuu ve he thong tiet do
mire de bieu hien de dang nhAn biet thong qua hoat
tinh phan cat cau tree amylose trong tinh bet, lam
mat mau thu& di* 12/KI (Nicholson, Chambliss,
1985). Gene amyQ dirge chen vao yang Multi
Cloning Site (MCS) cua pHT01 tao thanh pHT43amyQ. Sg kiem soat bieu hien tiet a-amylase dm
plasmid pHT43-amyQ trong B. subtilis 1012 va B.
subtilis WB800N Ichito sat.
VAT LIEU VA PFRIONG PHAP
Chiang vi sinh vat, plasmid va moi trierng nuoi cay
Chung E. coli OmniMAX (Invitrogen) dugc sir
dung trong cac bit& d6ng h6a. Hai chung B. subtilis
dugc sir dung
bieu hien protein mix tieu bing
chat cam img là IPTG: (i) chfing B. subtilis 1012
(Saito et al., 1979) bieu hien protease ngoai No; (ii)
chimg B. subtilis WB800N (Nguyen et al., 2011)
mang 'gene khang neomycine thay vi Cm va mang
(let bien bit hoat tam protease ngoai bao tir chung
WB800 (Wu et a/., 2002). Lru diem cila chimg
WB800N la it bieu hien protease ngoai bao, thuAn lgi
cho viec bieu hien cac protein dich dang tiet do sr
tich to tang din va giam sg they phan protein dich
ben cac protease ngoai bao. Plasmid pHT01 chira

promoter Pgrac va gene lad dugc sir dung lam
khung man de thiet ke plasmid pHT43-amyQ. Mau
chting am dugc thgc hien yeti plasmid pHT43
(Nguyen et al., 2011) c6 cAu true F ieng vai pHT01
nhung co them trinh tv tin hieu tiet cua a-amylase,
SamyQ. Te bit° dirge nuoi cAy tic tren mei tru&ng
Luria broth (LB) Or 37°C, khang sinh dugc them vac,
vai nOng 40 tuang img (amipicillin 100 lig/mid& vai
E. coli va Cm 10 lig/m1 doi yeti B. subtilis). Te bao
dugc trai ten mei tnrang thach chira 2% tinh bet
!thong tan de quan sat hoat tinh a-amylase.

a

Thiet ke plasmid
Doan gene amyQ (bao gem ca trinh to tiet cua
a-amylase) dugc thu nhAn to plasmid pKTH 10
(Palva, 1982) bang phin Ong PCR, sir dung clip mei
ON29,
5'GGCCATGGATCCATGATTCAAAAACGAAAG
CGGACAG-3'
va
ON42,
5'GGCCATGACGTCTTTCTGAACATAAATGGAG
328

ACGGAC-3'. Doan gene sau khi khuech dui c6 kich
thuOc 1545 by dugc cat vai 2 enzyme cat BamHI va
AatII, sau do not vai pHT01 ding dugc xir 13% yeti 2
enzyme cat tren. Vector tai to hop mai tao thinh

dirge dAt ten la pHT43-amyQ. San pham not dugc
bien nap vao cluing E. coli OmniMAX, sang loc va
kiem tra doan chen bang giai trinh ttr truck khi bien
nap vao hai chung B. subtilis 1012 va B. subtilis
WB800N.
Khan sat hotit tinh a-amylase tren dia tkach
Sau khi sang loc va kiem tra, IchuAn lac dun cea
hai chung B. subtilis 1012 va B. subtilis WB800N
chira plasmid pHT43-amyQ dugc nuoi cay ten dia
LB chira 10 1.1g/m1 Cm kh6ng be sung (0 mM IPTG)
hoAc c6 be sung IPTG tir 0,001, 0,01 va 0,1 mM, 2%
tinh bet khong tan. U trong 16 gib 6 37°C. Sir dung
dung dich 12/KI de xfic dinh yang phan giai tinh bet
do a-amylase (Nicholson, Chambliss, 1985). Chirng
am la pHT43 cam Ong v6i 0,1 mM IPTG. Thi
nghiem dugc leP lai 3 lan ten 3 Ichuin lac khic
nhau.
Cam Ong bieu hien a-amylase tren moi tritinig long
Hai chung B. subtilis 1012 va B. subtilis
WB800N dugc nuoi cAy trong mei truang LB yeti
khang sinh Cm 10 gg/ml Or 37°C va Vic de tic 250
vang/phet. Den khi to bac) vao gift pha log, ODA
dat 0,8 tien hanh cam Ong 6 4 neng de IPTG (0,
0,001, 0,01 va 0,1 mM). Mau dugc thu 6 0, 2, 4 va 6
h sau khi cam Ong. Thgc hien twang to yeti mau
chimg am la hai chimg B. subtilis 1012 va B. subtilis
WB800N chira plasmid pHT43, cam img va thu mau
am 6 6 h. Thi nghi'em dugc lAp lai 3 lAn ten 3 khuAn
lac khfic nhau.
Klein tra sg bieu hien bing SDS-PAGE

Mau sau khi thu a cac thai diem se dugc tach to
bao ra khoi mei truemg nuoi cAy bang ly tam (13000
yang/phut Or 4°C trong 10 phut). Protein c6 trong dich
nuoi cay dugc thu nhAn bang phuang phap tea TCA
(40%), yen ti le TCA va dich nuoi cay la 1:3, it trong
di 10 phut. Sau de ly tam (13000 vang/phiit It 4°C
trong 10 phut) thu tea va rim tea 2 Ian vOn aceton lanh.
Phai Icho a nhiet de phang, be sung ntrac, sample
buffer va bien tinh bing nhiet (95°C, 5 phut). Tien
hanh dien di SDS-PAGE tren gel polyacrylamide 12%
va tinh ti le a-amylase tren tang s6 protein tiet bing
phan mem AlphaEaseFC (Alpha Innotech)
( />fc.htm).


Top chi Gong nghe Sinh hoc 11(2): 327-332, 2013
KET QUA VA THAO LUAN
That Ice vector pHT43-amyQ
Gene amyQ duqc thu nhan bang phan 1mg PCR
vai cap mai dac hieu mang trinh tu nhan biet cita
enzyme cat giai han BamHI va AatII. San pham PCR
(1545 bp) va plasmid pHT01 dugc xtr ljr vai hai
enzyme BamHI va AatII, sau do tien hanh pban ung
not vai enzyme T4 ligase. Sin pham not dtrat bien
nap vao E. coli OmniMAX, sau do tien hanh sang
loc va giai trinh to nham xac dinh plasmid pHT43amyQ. Plasmid tai to hqp sau khi tach chiet tin chung
E. coli OmniMAX/pHT43-amyQ se duqc bien nap
vao B. subtilis 1012 va B. subtills WB800N.,Trai
tren mai truong LB-Cm nham sang lac nhang to bio
mang plasmid attic tieu. Cfic lchuan lac dac trong B.

subtilis 1012/ pHT43-amyQ va B. subtilis WB800N/
pHT43-amyQ se duqc sir dung cho cfic thi nghiem
khao sat a phan sau.
Khao sit ho#t tinh a-amylase
Ithuan lac clan B. subtilis 1012 va B. subtilis
WB800N mang plasmid pHT43 hoac pHT43-amyQ
duqc cham sang dia LB b6 sung Cm, 2% tinh bOt
khong tan, khong china chat cam an (Hinh 1B) hoac

pHT43
IPTG

0,1 mM

co chira chat cam img vai Ming dO IPTG la 0,001,
0,01 va 0,1 mM, it 16 gia a 37°C. VOng phan giai
tinh bOt hinh thanh do a-amylase duqc tiet ra moi
truang xung quanh phan cat cau trac amylose tong
tinh bOt, yang moil truerng thieu tinh bOt khong the
cing
bat mau vai dung dich 12/KI, yang phan
r6ng khi a-amylase duqc tiet tang nhieu. Ket qui
cho thay hai chung B. subtilis mang plasmid pHT43amyQ cho thrtmg kinh vOng phan giai tinh bqt 1(m
han nhieu (Binh 1B; 0 mM IPTG va Hinh 1C; 0,1
mM IPTG) so vai chimg am, B. subtilis mang
plasmid pHT43 (Hinh IA). Dieu nay chung to gene
amyQ tren pHT43-amyQ duqc bieu hien vtrqt mint
dual su dieu him dm promoter manh Pgrac. 6 cac
n6ng d6 IPTG 0,001 mM va 0,01 mM cho yang
phan giai lan han tnr&ng hqp 0 mM IPTG va nh6

han tnamg hqp 0,1 mM IPTG, nhtmg khong clang ke
(ket qui kh6ng the hien). So sanh giaa hai chung B.
subtilis, yang phan giai tinh bOt xung quanh khufin
lac B. subtilis WB800N (Hinh 1, Dtrai) hai cao han
so vai yang phan giai dm B. subtilis 1012 (Hinh 1,
Tren). Nhu vay, viec loai 136 protease ngoai bio 6 B.
subtilis WB800N cho phep a-amylase tiet ra m6i
trutmg co phan hai cao han so vai chung B. subtilis
1012.
pHT43-amyQ
0 mM

0,1 mM

1012

WB800N

A
Hinh 1. Khdo sat sty Neu hiOn oda a-amylase ten dia thach. B. subtilis 1012 tie B. subtills WB800N cht"ra plasmid pHT43-

kh8ng cam Ong (B) hoac co cam Ong vai IPTG b nOng dO 0,1 mM (C). MAu chirng am pHT43 duvc cam Ong vol 0,1
mM IPTG (A). Duteng ke mau trangli ban kinh yang phan

amyQ

Cam Ong bleu Ihre'n va SDS-PAGE
De danh gib mint d'6 bieu hien va tiet a-amylase
truck va sau khi cam ang, chung toi tien hanh nuoi

cay cua hai cluing B. subtilis 1012 va B. subtilis
WB800N china riasmid pHT43 hoac pHT43-amyQ
trong moi truong LB-Cm; cam ang vai 0,001, 0,01
va 0,1 mM IPTG va thu mitt sau 0, 2, 4, 6 gily cam
329


Phan Thi Phut:mg Trang et al.
Ong. Dich nuoi city china a-amylase ducc thu nhan
va xir ly nhu da pen a phanVat lieu — Phucmgphap.
Quan sat ket qua a mau cam ling vai Wing do
IPTG thap (0,001 va 0,01 mM) (Hinh 2), muc de
bieu hien protein muc tieu rat thap, ttrcmg ducmg vai
mau 'thong cam Cmg (Hinh 2; 0 h). Tuy nhi'e'n, khi
tang nOng de IPTG len 0,1 mM thi mac de bleu hien

A
kDa

lai tang clang Ice len den 30% protein tang se
(AlphaEaseFC). Trong khi cac matt twat cam ling
(Hinh 2; 0 h) Ichong nhan thay str hien diett .cda voch
protein muc tieu. Nhu vay su chenh loch ve muc de
bieu hien giva tnrac va sau cam ling là rat cao, hay
not cfich khfic, kha nang kiem sok bieu hien cita
promoter Pgrac la rat chat che.

B. subtilis 1012 / pHT43-amyQ
2h

M pHT43 0 h

4h

0 0001 0AI 01 0 0.001

200

0.01 01

6h
0 0.001 aol 0 1

100.

411.0"-ArnY°
.
.m0 iris. &MO Wilm.

30-

Iwo ..aa

B.

subtilis WB800N / pHT43-amyQ

2h
0

tiki 'amok

imaa

0001 001

4h
01

0

0001 001 0 I

6h
0

0001 001 01

am.

MAN

,inow -

Hinh 2. Kdt qud diOn di SDS-PAGE. M, thang protein PageRuler rm Unstained (Fermentas). Chung B. subtilis 1012 (A) va B.
subtilis WB800N (B) mang plasmid pHT43-amyQ cam Ong bang IPTG vai n6ng d0 0 mM (0), 0,001 mM (0,001), 0,01 mM
(0,01), 0,1 mM (0,1) khi ODA dot 0,8. Thu mau b cac thal dittrn 0 gib (0 h), 2 gib (2 h), 4 gig' (4 h), 6 gib, (6 h) deoi vai
pHT43-amyQ; mau cht:rng am thu b 6 gib, (pHT43).

So sinh sv bieu hien protein ngoai bao a hai

cluing B. subtilis, to thay, chi:mg B. subtilis WB800N
(Hinh 2B) cho vach protein muc tieu dam .han so vai
chung B. subtilis 1012 (Hinh 2A), re nhat a mau 6
gia sau cam ling. Chimg to, ltrung a-amylase tao ra
dr B. subtilis WB800N nhieu han so WA B. subtilis
1012. Ket qua nay hoin loan Oft hup vai hoot tinh
a-amylase bieu hien ten dla thong qua yang phan
giai ten dia tinh bet (Hinh 1). Tuy nhien, khi so sinh
330

tS, le a-amylase ten tong se' protein tiet thi B. subtilis
1012 lai cho ket qui tot han vai 30%
(AlphaEaseFC); trong khi, a B. subtilis WB800N thi
tjr le nay chi dot khoang 25% (AlphaEaseFC) mac du
Itrung protein muc tieu bieu hien nhieu han B.
subtilis 1012. Nhu vay viec loai be tam protease
ngoai bao a B. subtilis WB800N giftp halt che phan
cat protein muc tieu, nhtmg cling lam tich to nhieu
protein tiet khac trong mei tnrang.


Tgp chi Cong nghe Sinh hpc 11(2): 327-332, 2013
THAO LUAN
Nhting nghien ciru dau tien ve viec bieu hien
protein a-amylase trong B. subtilis &roc thuc hien
b6i Palva vao nam 1982. Gene amyQ tir B.
amyloliquefasciens &roc phan lap va .chen vao
plasmid pUB110 (Keggins et al., 1978), ket qua hinh
thanh plasmid pKTH10 cho kha nang bieu hien aamylase tang gap 2500 Ian so yeti chimg B. subtilis
Marburg va gap 5 Ian so v6i B. amyloliquefasciens

(Palva, 1982). Day la met plasmid sao.chep cuen
ton c6 tiem nang !thong on dinh ve eau trac
(Schumann, 2007), vai so luting ban sao là 50
plasmid/te bao va to cam irng. Met so nghien ciru
gan day
chimg toi cho thay, .viec Ai) dung
promoter Pgrac, vac) cac plasmid bieu hien tiet aamylase cho ket qua Luang ducmg v ri pKTH 10
(Phan et al., 2006). Tuy nhien, so vii pKTHIO c6
g6c tir pUB 110 thi pHT01 co ratting tru diem. vuort
trei: (i) co the sao chep theta, giup on dinh ye mat
cau trite trong to bao, (ii) so luting ban sao 4 — 6
plasmid/te bao, hart the bleu hien nen va (iii) dac
diem cam img bang IPTG cho phep kiem sok tot
viec bieu hien protein. muc tieu. \Teri uu diem nay,
Pgrac da cho thay tiem nang irng dung hieu qua
trong viec dieu hoa bieu hien protein tai to hop, nhat
la nhimg protein gay dee cho to bao va gidp bieu
hien protein muc tieu i nhang thOi diem
mc;ng muon.
Viec sir dung chimg (let Wen. B. subtilis
WB800N cho phep protein muc tieu bieu hien nhieu
hcm trong m8i tnrang nuoi cay do it bi anh hu6ng
boi protease ngoai bao, cho thay hieu qua sir dung
chimg dot bien nay. Tuy nhien, viec giam tiet
cac protein khac (ngoai protein muc tieu) ra moi
twang coy nglira rat quan tong ,khi tinh sach sau
bieu hien. Do d6, met hart che ctia B. subtilis
WB800N la cho phep tich to nhieu protein ngoai bao
khac, dieu nay lam giam phan tram protein muc tieu
teen tong so protein tiet. Mac di' vay, vii kha Jiang

bleu hien wet tot len den 25% tong protein ngoai bao
thi B. subtilis WB800N se la met cluing chit tiem
nang trong viec bieu hien protein muc tieu du6i clang
tiet, dac biet la nhang protein de bi phan cat 136i
protease ngoai bao. Trong khi do, cac chung bieu
hien protease ngoai bao nhu B. subtilis 1012 lai thich
hop cho viec bieu hien cac protein tai to hgp tuung
doi ben v6i cac protease nay.
KET LUAN
Tir ket qua kith) sat hoat tinh a-amylase tren dia

thach va su bieu hien trong,moi trutmg long the hien
tren SDS-PAGE, CO the ket luan nhu sau. Plasmid
pHT01 chira promoter Pgrac là met he vector bieu
hien manh cho B. subtilis giup dieu hoa bieu hien
gene muc tieu met cach hieu qui len den han 25%
tong protein flit. Plasmid pHT01 c6 the sir dung
cling v6i chung B. subtilis WB800N trong viec bieu
hien tiet protein mix tieu, (lac biet phi' hop cho
nhitng protein bi thity phan bori protease ngoai bat)
dm B. subtilis.

a

Ltd cam .ant Nghin
e cau nay dupy tai trpr
phat trien khoa hpc va corm nghe quoc gia
(NAFOSTED) trong de tai and so 106.16-2011.80.
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STUDY ON SECRETIONAL EXPRESSION OF a-AMYLASE REPORTER IN BACILLUS
SUBTILIS WB800N USING VECTOR pHT01
Phan Thi Phuong Trang, Nguyen Hoai Nam, Tran Linh Thuoc, Nguyen Due Hoangs
University of Science, Vietnam National University Ho Chi Minh City

SUMMARY
The appearance of the first comericial plasmid pHT01 promotes the study for inducible over-expression of
recombinant protein in Bacillus subtilis (B. subtilis). Most of researches published relating to this plasmid used
B. subtilis 168 derivatives, such as 1012 strain, but not with WB800N — eight-fold protease-deficient strain. To
explore the possibility to use plasmid pHT01 with B. subtilis WB800N for secretional over-expression of

recombinant protein, we used secretional reporter protein, a-amylase encoded by amyQ from Bacillus
amyloliquefasciens. Plasmid pHT43-amyQ was constructed from pHT01 and used to investigate the secretional
expression of a-amylase in both B. subtilis 1012 and B. subtilis WB800N. The results showed that a-amylase
could be produced efficiently in both strains, in which the amount of the reporter from WB800N was higher
than that from 1012. This result suggests that plasmid pHT01 can be used with B. subtilis WB800N for
secretional expression of recombinant protein.
Keywords: amyQ, Bacillus subtilis, plasmid pHT, pHT01, promoter Pgrac, WB800N

* Author for correspondce: E-mail:

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