Tgp chi Cdngnghe Smh hoc 11(1). 131-138, 2013
D A N H G I A SU B I E U H I E N G E N B E T A - L A C T A M A S E O C A C C H U N G N.4.M M E N PICHIA PASTORIS T A I T O H O P S I N H I N T E R L E U K l N - 2 \ A A M Y L A S E B A N G K Y T H U A T REAL-TLME P C R
Le Thj Lan Anh, TrJin Nhat Anh, Than Van Minh, Nguyen Hdng Thanh, Dirong Cam Thiiy, Le Thi Thu Hong, Trirong Nam Hai
Vien Cong nghe sinh hoc, Vien Hdn ldm Khoa hoc va Cong nghe Viet Nam TOM T A T
Nam men Pichia pasioris (P pastoris) duoc su dung pho bien Irong san xuat cac protein ngoai Iai co nguon gdc khac nhau, Tuy nhien ca che phan Xu ddi vdi qua trinh bieu hien gen ngoai lai uong chung nam men nay van chua diroc ngliien cuu day dii Nhirng phan tich transcnplome dira Iren ky thuat microarray ddi voi cac chung nam men P. pasioris tai Id hop da chi ra cd sir khac hiet ve miic do phien ma cua gen beta-1 acta rnase (hta) giQa chiing ddi chimg va cac chiing mang gen ngoai lai amylase/interieukin-2 (Amy/IL-2) Trong nghien cuu nay, chiing Idi sii dung ky thuat real-time PCR de Iam sang to moi quan he giua su bieu hien cua gen bia va gen ngoai lai Amy/IL-2 trong cac chung tai Id hop a muc do phien ma Phan tich real-time PCR dircrc thuc hien vdi cac mau nghien ciiu ciia chung P pasioris ddi chirng va cac chiing sinh Idng hop (Amy/IL-2) tai ihdi diem 0, 6, 12, 24, va 48 h sau cam img methanol Ket qua phan tich real-Ume PCR cho thay cd su khac biet Idn khi so sanh giua chung mang gen va chung ddi chung o tat ca cac thdi diem nghien ciru, tuy nhien khong cd su khac biet dang ke giira hai chun^ mang gen Su phien ma cua gen bia Irong cac chung mang gen so vdi chung doi chirng dat gia In cao nhat gap hon 45 lan tai then diem 12 h sau cam img methanol Hon the nira, ket qua phan tich cdn cho thay cd sir kliac biet nho khi so sanh theo thong sd thdi gian trong moi chung nghien cini Cu the, su khac biet d cac thdi diem nghien ciiu trong chiing ddi chimg Idn hon 2 lan, ben canh dd trong chung IL- 2 cung CO sir khac biet Idn hmi 2 ISn giua thdi diem 24 va 48 h, Cac so sanh lai cac thai diem con lai deu khong CO sir khac biet Idn va deu eo gia tn nho hon 2
TU khoa: An Iranscnplomic
hieu hien gen. -.'rleukui-2. Puhui pas PCR.
MO DAU
Nam men Pichia pasioris (P. Pasioris) duac sir dung phd bien trong nghien ciiu bieu hien gen ngoai Iai (Cereghino and Gregg, 2000, Gregg el al, 2000). Vdi chiing nam men nay, nhieu prolem ngoai lai duoc tao ra vdi ham luong ldn cd the dal tren 10 g/I, tuy nhien mdt sd protein lai duoc bieu hien d mirc dp th4p (dudi I mg/I), hoac khdng xac dinh duoc Su bieu hien protein ngoai lai cd the gay ra cac dap irng ciia te bao chii va cd the Iam thay ddi qua trinh trao ddi chSt cua te bao dan den su khdng ben cua gen ngoai lai, pha vd cau triic ribosome, uc che smh trudng tham chi pha vd te bao va nhu v$y nd cd the anh huang den kha nang tdng hgp protein ngoai lai ciia te bao. De nang cao kha nSng bieu hien ciia protein, mdt sd phuong phap da dupc sir dung nhu: thay the ma bd ba cho phii hop. bieu hien ddng thdi vdi chaperone (Chang et al, 2006) hoac vdi cac protein khac (Damasceno el al, It^tft; Zhang el at., 2006), gay dot bien mot sd gen ciia chung bieu hien (Zhao et at., 2008). tdi
uu dieu kien nudi cay (Zhang et al. 2007, Gurramkonda et al, 2010), , , Cac dieu kien duoc thiet lap cd the dugc ap dung de Iam Iang su bieu hien cua mdt sd protein tai td hop khac. tuy nhien khdng phai tat ca cac trudng hgp deu thanh cdng do nhung hieu biet ve di truyen phan Iti: va qua Irinh ly hda trong chung nam men nay van cdn han che.
Nhimg ket qua phan tich so sanh he transcnplome ciia chung P pastoris ddi chimg, chiJng sinh tdng hgp intedeukin-2 (IL-2). ehung sinh tdng hop amylase (Amy) sir dung YEAST_2 array, bao gdm khoang 12 000 bg mau dd (probeset) dac hieu cho cac gen trong he gen cua Sacchciromyces cerevisiae va Schizosaccharoinyces pomhe cho thay cd 6 gen cd su khac biet trong bieu hien gen giCra cac ehung nghien cuu la MF(ALPHA>t (pheromone alpha factor), bia (beta-tactama.'ie). RPLIO (60S ribosomal prolem LIO), GAL2 IGalaclosc permease), RPB2 (RNA polymerase II second largest subunit BI50), PTK2 (Putative serme'lhreomnc protein kinase) (Duong el at. 2012) Trong dd, gen
l.tl
Le Thi Lan Anh el al bia cd \ai trd pha vd vdng P-lactam ctia cac chat
khang sinh nhu penicillins, cephamycin va carbapenems Gen hla khdng cd mat trong chiing P.
pasioris gdc ban dau, nhimg trong vector bieu hien pPlC9, gen nay dugc thiet ke cho muc dich nhan ddng cua vector nay trong te bao vi khuan va do do dugc dua vao cac chiing nam men P. pastoris trong qua trinh tao chiing tai td hop, Gho den nay chua cd nghien cim nao Ihdng bao ve mdi lien he giiJa sy bieu hien ciia gen ngoai Iai va gen hla trong cac he bieu hien,
Trong nghien ciru nay. chung tdi hen hanh thi nghiem danh gia va phan tich su bieu hien cua gen bia trong cac chung nghien cuu bang ky thuat real- time PGR vdi cap mdi dac hieu, Vdi tinh nang uu Viet ciia Ky thuat real-time PCR trong phan tich su bieu hien gen nhu dd nhay cao hon, dd chinh xac Idn hon va cd the thyc hien vdi sd luong mau Idn hem se giup danh gia va phan tich chinh xac sy bieu hien ciia gen bia, tir dd cd nhung dinh hudng cho nhtmg nghien ciru tiep Iheo nham nang cao miic do bieu hien cua gen ngoai lai trong chiing nam men P pasioris tai td hop,
VAT LIEU VA PHU'ONG PHAP
\ ' a t lieu
Cac chung P pasioris SMDI168 Iai td hgp da lich hgp pPIC9 (P pastoris pPiC: chting ddi chimg - control), P pastoris pPIC9Amy (ehung Amy), P pasioris pPIC9IL-2 (chung IL-2) dyoc su dung cho nghien ciru.
Trinh ty mdi dd nhan gen IPPl, 6/ij.Fw-IPPL 5-TAGGGTGGCTTCCGTCAG-3'; Rv-IPPI: 5 - TCCCTCATCCAACAAAGCCATAAG-3', Fw-bla' 5'-CGCCTTGATGGTrGGGAAGC-3', Rv-bia 5"- ATAGT ACGATACGGGAGGGCTT AC-3',
Cac mdl trudng nudi cay: MD (1.34% YNB.
4-I0"Vo bioun, 1% Dexttos), BMGY (lOmM Potassium phosphate pH6, 1,34% YNB, 4,10"-%
biotm, 1% methanol)
Hda chat cho real-time PCR' Bd kit tach chiet mRNA Idng sd ctia hang QiAGen (Due). Bd kit Real-time PCR lighl cycler 2.0 one step cua hang Roche (Ddc)
Cac hda chat, enzym dung trong sinh hoc phan td duoc mua tir hang Fermentas (My) gdm DEPC, DNasel,
P h i n m g phap nghien e u u ten men le bdo ndm men tdi to hffp
Nudi cay len men cac chung nam men de thu m l u tach chiel RNA theo phuong phap da trinh bay trudc day (Duong el al., 2012) Mdt khuan lac chiing nam men tai td hgp duac nudi cay trong moi trudng BMGY Idng, lac 28''C qua dem den OD^o - 6. Chuyen dich nudi cay sang mdi trudng BMGY mdl sao cho OD ban dau ~ 0,1, nudi cay d 2 8 ^ , 200 vdng/phiit cho tdi khi ODaog - 13-16, cam iing bang methanol den ndng do cudi ciing la !%, Sau 12 h tiep tuc cam ung methanol lan 2 ndng do nhu cam ling lan I. Mau te bao nam men va dich protein trong mdi trudng nudi cay a cac thdi diem 0, 6, 12, 24. 48 h sau cam iing bang methanol.
Tdch chiet vd tinb sgch mau RNA
Mau RNA tdng so cua te bao nam men duoc tach chiet dya tren kit tach chiel ciia Qiagen (RNeasy Mini Kit), frong dd te bao dugc pha theo phirong phap dung giai enzyme Mau RNA sau tach chiet va tinh sach dugc kiem fra fren gel agarose, dinh lugng va xac dinh dd sach bang may do quang pho NanoDrop, Sau khi do ndng do, mau duac pha loang den ndng do IOO ng/pl va dugegiiJ d -80"C cho den khi tien hanh thi nghiem real-time PCR, Tong hffp phien md ngug'c
Real-time PCR su dung chat phat huynh quang duac tien hanh dya fren bd Kit LightCycler'E' RNA Master SYBR Green 1 Real-time PCR vdi moi cSp mdl dugc thuc hien frong timg dng rieng biet, Phan img duoc tien hanh vdi cac thanh phan nhu mdi, Mn(0AC)2 , PCR master mix va mRNA khudn, Doi chimg am la mau khdng cd mRNA tdng sd,
Chugng frinh chay real-time PCR mgl budc gom 2 phan ung'
Phan img phien ma ngugc dugc tien hanh o 6 r c trong 30 phiit
Phan ling khuech dai cDNA bao gdm nhung budc sau: i) xu ly nhiet vdi mau phan img d 95''C frong 2 phut, ii) 40 chu ky khuech dai nhu sau:
bien tinh cDNA d 95''C trong 10 s, g5n moi cr nhiet do gan mdi SS^C trong vdng 30 s va khuech dai cDNA d 72''C trong vdng 30 s va iii) phan iing keo dai frong 5 phut. Real-time PCR dugc thyc hien fren may LightCycler cua Roche version 2.0 va sd lieu dugc phan tich bSng phSn mhm Lighl Cycler 4.0
Tap chi Cong nghe Sinh hgc 11(1): 131-138,2013 So sdnh tirtrng doi ham Iirffng RNA cua gen dich trong cdc mau nghien cuu
Phan ling khuech dai bang real-time PCR diln ra Iheo cap sd nhan, do do cac mau cd chu ky ngudng chenh nhau I don vi luong duong vdi su khac biet khoang 2 lan ve ham lugng RNA ciia gen dich, Trong thi nghiem cua ehiing Idi, viec so sanh lugng RNA trong cac mau thi nghiem dugc hen hanh phuang phap dinh lugng tuong ddi dua fren don vi khdi lugng Ty Ie lugng RNA giira mau thu va mau ddi chiing duoc tinh theo cdng Ihiic. Ty Ie mau thu/ddi chiing = 2'^'"^'-*^"^' , Trong dd, Ct(C) la chu ky ngudng cua mau ddi chiing, va Ct(T) la chu ky ngudng ciia mau thi nghiem.
KET QUA
Tach chiet mRNA ciia chiing nam men P. pasioris tai to hop.
Ket qua phan tich real-time PCR phu thudc rat nhieu vao chat lugng cua mau mRNA khudn llllumina, 2010). Ham lugng RNA cua cac mau dugc tach chiet dat tir lOOng/yl den 1291 ng/pl vdi do sach dat yeu cau (ty !e OD26o/OD2m} ciia tat ca cac mau cao xap xi 2), Ket qua nay chung td cac mau RNA khdng bi nhiem prolein cdng nhu phenol Irong qua trinh tach chiet. Them vao dd, kel qua dien di
tren gel agarose cho thay cd cac bang 5S, 188 va 28S rd net va khdng bi phan c5t,
Toi tru dieu kien cho real-time PCR Ben canh ndng do RNA khudn, ndng do mdi, nhiet do va thdi gian gan mdi,.,, ciing anh hudng den ket qua phan tich real-time PCR. Trong dd. dya vao trinh Ur ciia doan mdi da dugc thiet ke, cd the suy doan duac nhiet do va thdi gian gan mdi phii hop, Vi vay, ndng do khudn RNA va ndng do mdi sii dung cho phan ting can dugc tdi uu de han che su lao thanh cac san pham khdng dac hieu,
Trong ihi nghiem dai ndng do 1, 50, 250 va 500 ng/pl cua RNA khudn vdi cap mdi ciia gen bia dugc khao sal, Ket qua dien di cho thay d ndng do RNA khudn la I ng/pl, kho quan sat thay sy cd mat cua san pham gen bia trong khi vdi cac ndng do cdn lai deu tao san pham cd kfch thudc 302 bp dac hieu cho gen bia (Hinh IA), Vi vay, ndng dd RNA khudn la 50 ng/jil dugc lua chgn cho thi nghiem ldi uu ndng do mdl,
Cac ndng dp mdi da duoc su dung de danh t;ia 0,_2, 0,4, 0,8, I va 1,2 mM. K a qua dien di cho ihay, ndng do mdi trong khoang 0,4 -1,2 mM, deu cho ra san pham cd kich thudc 302 bp theo tinh loan, Vi vay, ndng dd mdi su dung cho phan img real-lime PCR la 0.4 mM
1 2 3 4 M
Hinh 1. San pham ciia phan irng real-time PCR cho tdi u'u nong do Khuon mRNA va nong dp moi, 2A, 1 - 4 Nong dp mRNA tuong img la 1, 50, 250, 500 ngi [i\; 2B. 1 - 6 Nong dg m6j luong irng la 0,2, 0,4, 0,8; 1 va 1,2 mM M Thang chuan DNA 100 bp (fermentas).
Real-time PCR ve bieu hien ciia gen noi chuan IPPI (ien ngi chuan (reference gene hay internal slandard) la cac gen cd sy bieu hien phien ma khdng ddi d cac trang thai td bao khac nhau, dugc Sli dyne trong real-time PCR nham dinh lugng tuong ddi sy bieu hien cua gen dich de tir dd Ioai bd sai sd frong qua Irinh Iam thi nghiem Do vay, tim gen ngi chuan phii hgp cho phan irng real-lime PCR
la can thiet, Sau khi iham khao mdt sd gen ndi chuSn HPRT PBGD. GAPDH. G6PDH, RPLIO.
IPPI. ALG9, ARG4. UBC6, TAFIO, etc cho thiy chi cd gen IPPI (cytoplasmic inorganic pyrophosphatase) la phu hgp vdi yeu cau trong nghien ciiu nay.
Real-time PCR duoc tien hanh vdi gen ndi chuan IPPI a cac chung control, Amy va IL-2 lai 133
L e T h i Lan Anh el al cac thdi diem 0, 6, 12, 24, va 48 h (Hinh 2). Thi
nghiem dugc lap lai 2 Ian vdi mau RNA dugc tach chi^t tir cac ISn Ien men khac nhau. Ket qua cho thay rang sy bieu hien ciia gen ndi chuan khdng dugc nhu mong mudn (Bang 1). Cu the la, khi so sanh tai Ihdi dilm 24 h so vdi 0 h, d chung ddi chimg va chimg
Amy, sy bilu hien gen IPPI giam khoang 5 lan va khoang 10 lln tai thai diem 48 h. Ben canh do, d chung IL-2, tai thdi dilm 24 h va_48 h, sy bieu hien cua gen IPPI cung giam hon 10 Ian so vdi 0 h. Nhu vay, gen nay khdng phu hgp vdi tieu chuan ctia gen ndi chuan.
i 23
— 25 C3-12H-7
— 31 l3-24H-r 8 C3-6H-ie
— 20 A3-i2H-7 26 C3-24H-7 32- 13-4 3H-7
— 3 A3-6H-16 9 C3-12H-16 15 13-24H-26 21 fl3-24H-7 27 C 3-4 EH-7
4 A3-12H-16
•— 10 C3-24H-16 16 13-48H-16
— 22 a.3-4BH-7 29 I3-0H-7
- 5 A3-24H-16 11 C3-46H-16
— 17 DC-7 23 C3-0H-7 29 I3-&H-7
6 A3-4eH.16
— 12 I3-DH-1S
— 24 C3-SH-7
37 33 39 40
Hinh 2. Bi6u do real-time PCR khuech dai cac gen cua cac chiing nar c Ihoi diem khac nhau
Bang 1 . K^t qua real-time PCR dmh lugng tuong doi s u bieu hien ciia gen IPP1 bang p h u o n g phap d y a tren do'n V! khoi lu^ng Su bieu hien cua cac chung nghien cuu t^i cac thoi diem d u g c dinh lugng so vdi s u bieu hign cua chung control lai tlnoi diem 0 gio'. Thi nghiem duoc lap lai 2 lan
Mau ttr so 1-16 la gen ndi chuan IPPI Mau sd I: ddi chiing am khdng cd RNA long sd. Mau 2-6 tuong ling cac mau phan img ciia chiing Amylase tai 0, 6, 12, 24 va 48 gid, Mau 7-11 tuang ting la cac mau phan iing ciia chung control tai 0, 6, 12, 24 va 48 h. MSu 12-16 tuong img la cac mau phan ung ctia chimg mterIeukm-2 lai 0, 6, 12, 24 va 48 h.
M a u tir so 17-32 la gen dich bia. M l u sd 17:
ddi chiing am khdng cd RNA long sd, Mau 18-22
tuong ling cac mau phan iing ciia chiing Amylase tai 0, 6, 12, 24 va 48 gig M l u 23-27 frrgng ung la cac mau phan ung cua chung control tai 0, 6, 12, 24 va 48 h. Mau 28-32 luong iing la cac mSu phan iing ciia chiing interIeukin-2 tai 0, 6, 12, 24 va 48 h.
Ket qua real-time P C R ve bieu hien cua gen bia Real-time PCR dugc tien hanh khdng cd gen noi chuan va chuan hda cac dieu kien thi nghiem de loai bgt cac sai sd he thdng, Su bieu hien cua gen dich
Tap chi Cong nghe Smh hoc I l l i ) : 131-138.2013
dugc danh gia dua tren gia tri chu ky nguong Ct theo su khac biet ion trong chung .Am\ o cac thoi diem dcm\i khoi luong. nghien ciru {ty le khac biet nho hon 2) (Bang 31
Ket qua chu ky nguong cua cac mau trong mot lin
thinghiemduocthShien6hinh2.Thinghiemduoclap ° * " ' " ° S'a tri chu ky ngucmg cua 3 chung lai 3 ISn. Dua vao chu ky nguong cua 3 ijn' thi nghiem " E " " ™ ' * " " ' ^ ' * < " " l ' ™ '^'"' 1 " ^ "'"^ I™ men de de tinh chu ky nguong Uung binh (Bang 2). ^'"^ 8,'^ ^'?'' ''^™8 ctia su bieu hien cUa gen bia theo yeu to chung cho thay co su khac biet giira Phan tich su bieu hien cua gen hla o 3 chung chung d6i chimg voi chung Amy va IL-2, Tuv nhien nghien cuu theo thong so then gian cho thay co su i;|,ang co sit khac biet co y nghia khi so sdnh aiua khac biet. O Chung doi chimg. khi so sanh thoi diem 0 chting Amy xa IL-2. Su khac biet cua Ben hla aUa h VOI cac Ihoi diem 6, 12, 48 h, then diem 48 h va cac chung mang sen (Amy'IL-2l >oi chune d6i chtaB thoi diem con Iai cho thSy co su khac biet Ion hon 2 , j „ g ijn iheo thoi gian dat gia ui Ion nhfl hon 4.> i i n Ian, ben canh do cung thay su khac biet trong chung „ ,hf„ jiem 12 h la sau doliiam dSn o cac thoi diem IL-2 giita then diem 24 h va 48 h. Tuy nhien. khong co 24 h va 48 h cua qua trinh Ien men (Bang 41 Bang 2: Gia tn Ct trung binh cua gen bia a cac chung p paslons ngnien cuu a cac thoi di^m khac nhau cua qua trinh len men Gia tn cua mfli m i u auoc tmh la gia tn lrung bmh cua 3 lan lap lai Ihi nghiem
T h o i diem Oh 6 h I Z h 24 h 43 h
lay m lu phar 1 tich Control 23 65 24 85 24 85 '24 10 22 40
Gia Amy 19 50 20 05 19,35 19 20 20 05
t n Ct t r u n g binh IL-2 19 35 19 45 1 9 3 5 18 80 20 05 Bang 3. Ty le khac biet cua gen bla Irong cac c | p so sanh theo Ihdi gian ciia cung moi chung P pastons nghien cuu lai diem k h ^ nhau cua qua trinh len men theo giS In chu ky nguo-ng trung bmh Gia tn cua moi thai dien dune so sanh voi t;
Ihoi diem con lai Irong cung 1 chiing NhO'ng gia In duoc danh dau mau nau chi ra s y khac biel > 2 lan trong cap so sanh
Bang 4. Ty 1$ khac biet cua gen bla trong cac cap so sanh 6 cac chung P pastoris nghien cuu a cac thoi diem khac nhau cua qud Innh len men theo gia tn chu ky nguong lrung bmh So sanh su bieu hien ciia lung cap chung nghien cuu trong cung mpt th^i diem lay mau
Thd-i aiim l^y m l u phan tich
12 h 24 h 48 h
Amyfcontrol 17 8 27 9 45 3 29 9
Ty le khac biet gi
!L2/control 1 9 7 42 2 45 3 39 4
Le Thi Lan Anh et al.
KET QUA VA THAO LUAN
Real-time PCR va microarray la hai ky thuat bd sung cho nhau va dugc su dung rdng rai cho nghien cihi sir bieu hien cua gen Irong sinh hoc hien dai Sau khi xac dinh su bieu hien cua nhieu gen dua vao ket qua microarray, mdt sd gen quan tam cd the duoc sang Ipc va dugc danh gia vdi do chinh xac ldn hon, do phan giai tdt hgn va hieu qua hon vdi sd mau nhieu hon bang real-time PCR {Der\eau\ el al., 2010). Su phu hop cua ket qua thu dirge tu DNA microarray va real-time RT-PCR la rat quan trgng, Tuy nhien, thyc te nghien ciiu da chi ra trong mdt vai trudng hgp cCing tim thay sir khac nhau gtira ket qua microarray va real-time PCR Do dd, su bieu hien khac biet dugc xac dinh bdi microarray can phai duoc xac nhan lai bang phuang phap real-time PCR trudc khi cd cac nghien cuu tiep theo
Dinh luimg su bieu hien cua gen bang ky thuat real-time PCR ddi hoi mdt su chuan hda cac dii heu thich hop dc giam tdi da sai khac tao ra do ky thuat (Vandesompele ei at 2002) Cach tiep can duoc su dung phd bien nhat de phan tich su bieu hien ctia gen la dua vao cac gen ndi chuan, dd la nhUng gen cd mirc dd bieu hien dn dinh trong cac trang thai khac nhau cua ie bao (Nolan el al. 2006) Tuy nhien, viec tim dugc mdt gen ndi chuan tdt, phii hop la rat khd va thuc le la khdng phai khdng cd su thay ddi irong su bieu hien eua gen ndi chuan (Radonic el at. 2004).
Tren thuc te, mdt sd nghien cuu da tien hanh real-time PCR ma khdng diing gen ndi chuan {Nevoigt el al.
2006: Sun ei at, 2012, Bansal er «/., 2012) Hon nua.
he gen cua P. pastoris mdi dugc cdng bd each day khdng lau (De Schurter et a l , 2009), cac nghien cuu chuyen sau ve su bieu hien phien ma ciia chung nam men nay cdn han che Vi vay, chiing tdi khdng su dung gen ndi chuan trong nghien cuu sir bieu hien cua gen hla d cac chung nam men P pastoris tai td hgp ma cd gang chuan hda cac dieu kien thi nghiem real- time PCR de loai bd tdi da nhimg sai khac he thdng.
Gen bla ma hda cho enzyme be I a-lactamase thudng thay d vi khuan dugc su dung Iam dau chuan chpn Ipc trong thiet ke vector pPIC9 diing cho bieu hien gen ngoat Iai g nam men. Nhu vay, gen nay se dupc dua vao nam men trong qua trinh tao chiing tai td hpp Khac biet gen nay dupc quan sat thay giija chung ddi chimg \ a hai chung mang gen tai td hgp (AMY/IL-2) Trong kgl qua thu nghiem bang microarray tai ca 3 thdi diem 0, 24 va 48 h trong chung ddi chimg cd ty Ie khac biet gap khoang 15 ian tuy nhien khdng cd su bieu hien thay ddi theo thdi
gian trong cac chiing nghien cim (Duong el al, 2012).
Ket qua real-time PCR danh gia su bieu hien cua gen hla trong chiing ddi chirng so vgi cac chiing mang gen dat gia tn cao nhat gap hon 45 lan tai thdi diem 12 gid sau khi cam iing methanol (Bang 4). Nhu vay, ket qua trong phan tich real-time PCR ciing phii hpp vdi ket qua ciia phan tfch microarray, tuy nhien do dd chinh xac va dac thd cua mdi phucmg phap ciing vdi viec md rgng them mdt sd thdi diem lay mau ma ket qua phan tich \ e gen bla bang ky thuat real-time PCR cho thay cd su thay doi ldn hgn khi so sanh chiing mang gen va khdng mang gen va ben canh dd cOng cd sij thay ddi theo thdi gian so vdi thi nghiem microarray trudc day Ket qua real-time PCR va microarray cho thay sir bieu hien cua gen nay dugc (ang cao d eac chiing mang gen so vdi chiing ddi chiing Dieu nay co the giai thich la do sd lupng ban sao gen nay trong cac chting mang gen nhieu hon so vgi chiing ddi chiing hoac cd mdl lien quan giira gene bla va su bieu hien cua cac gen .Amy va IL-2
Viec gen bla bieu hien cao hon d cae chung mang gen ngoai lai va viec mirc dp bieu hien ciia gen nay ludn dao ddng xung quanh mdt gia tri trung binh, ggi y rang san pham cua gen nay cd the ddng vai trd dieu hda sir bieu hien gen ngoai Iai, qua do bao ve cg the vai chu P. pastoris. Nguyen nhan va co che ciia hien tupng nay cdn chua duoc lam ro, can co nhiing nghien ciru chuyen sau hem. Viec phat hien ra tac ddng dieu hda cua gen hla cd the giiip hieu ro hon ve qua trinh bieu hien gen ngoai Iai trong chiing nam men P pastoris vdi vector bieu hien dugc sir dung pPIC9 nham nang cao mirc dg bieu hien cua gen ngoai lai.
KET L U A N
Muc dd bieu hien cua gen bla d cac chiing P pa.Hloris mang gen Amy/lL-2 cao hon nhieu lan so vdi chiing ddi chiing khdng mang gen, tuy nhien khdng cd sir khac biet dang ke khi so sanh giua 2 chiing mang gen vdi nhau, Ben canh dd nghien ciru cdn cho thay cd su khac biet nhd ve muc dp bieu hien ciia gen bla trong cac chung nghien cim d cae thdi diem Ien men Ket qua phan tich su bieu hien gen nay gdp phan xac nhan lai rang trong qua trinh phan tich bieu hien gen dua vao microarray, viec khang dinh Iai ket qua bieu hien dua vao mdt phan tich ddc lap khac la rat can thiet,
Ldi cam (ra:_C6ng trinh dugc thuc hien nha kinh phi va Irang thiet bi ctia Phong ihi nghiem trgng diem 136
Tap chi Cong nghe Smh hgc 11(1)- 131-138,2013 Cong nghe gen Vien Cong nghe sinh hoc, Vien Hdn ldm Khoa hoc va Cong nghe Viet Nam
TAI LIEU THAM KHAO
Absolute Quanlincaiion of gen expression using SYBR Green in the Eco™ Real-Time PCR system (2010) Illumina Bansal R. Mamidala P. Mian MA. Mittapalh 0, Michel AP (2012) Validation of reference genes for gene expression studies in Aphis glycines (Hemiptera: Aphididae). J Econ Eniomol 105: 1432-1438
Gurramkonda C, Polez S. Skoko N. Adnan A. Gabel T.
Chugh D, Swaminathan S, Khanna N, Tisminetzky S, Rinas U (2010) Apphcation of simple fed-batch technique to high-level secretory production of insulin precursor using Pichia pastoris with subsequent purification and conversion to human insulin Microh Celt Fact 9 (31) Duong C T. TTH Le, TN Nguyen, HT Nguyen. TLA Le, NH Truong (2012) Mi era array-based transcnplome analysis of recombinant Pichia pasioiis sirains overex press mg alpha- amylase and inierleukin-2. IJBBB 2 1 -11
Chen C, Snedecor B, Nishihara JC, Joly JC, McFarland N, Andersen DC, Batlersby JE, Champion KM (2004) High level aecumulauon of a rccombinanl aniihody fragment in the periplasm o\' Escherictiui coli requires a triple mutant IdegP pre spr) host strain, Biolechnol Biocng K5 463-474 De Schuner K Lin YC. Ticls P. Van Hcckc A, Glinka S.
Weber-Lehmann J, Rouzc P. Van de Peer Y, Callewaert N (2009) Genome sequence of ihe recombinant prolein production host/•«/)(«/jmrwfj Nai Biolechnol 21 561-566 Fernandez JM, Hoeiller JP (1999) Gen expression System Acedamic press' 158-191
Zhao ML, Xue C. Wang Y. Yao XQ. Liu ZM (2008) Increasing the cell viability and heterologous prolein expression of Pichia pastoris mutant dcllcitnl in PMRI gene by culture condiiioii optimi/aiion Appl Mmohiol Bialeclmol HI-235-24\
Han MJ. Lee SY (2003) Proleome profiling and ils use in metabolic and cellular cnmneerini; Pioieomics 3 2.^17- 2324
Han Ml. Jeong KJ, Y.xi JS. Lec S'l" (2003) Engineering Escherichia coli (or increased productivity of serine-rich proteins based on proleome piotlling Appl Environ
Miuohioie"-) 5772-57SI
Cereghino JL, (icgg IM (201)0) Meierokigous prulcin expression in the mciliyloirophic yeasi I'ichia pastoris FEMS Microbiol Rci 24 45-66
Cregy JM, Cereghino JL. Shi J, Higgins DR (2000) Reeombinanl proicin expression in Pichia pasioiis Moi Biolechnol 16, 23-52.
Damasceno LM. Anderson K.A. Ritier G. Cregg JM. Old U , Batt CA (2007) Coove rex press ion of chaperones for enhanced secretion of a single-chain anIibod\ fragment in Pichia pastons Appl ypicrobiol Biolechnol 14 3SI-389 Klatte M. Bauer P (2009) Accurate Real-ume re\erse transcnplion quaniiiaiive PCR Methods Mot Biot 479-61-11.
Nevoigi E. Kohnke J. Fischer CR, Alper H. Stahl U.
Stephanopoulos G (2006) Engineering of promoter replacement cassettes for fine-tuning of gene evpression in Saccharoimces cciesisiae Appl Envnon Microbiol 72 5266-73
Radonic A Thuike S. \Iacka\ l.\L Landl O. Sieqen W.
Nilsche A (2004) Guidehne to reference gene seleclion for quanlitalive Real-time PCR Biotliem Bioplns Res
313:856-862-
Rockett JC (2003) To confirm or not to confirm (microarray daia)—lliai is ihe question Drug Disi ov Toda\
8,343,
Chang SW. Lee GC. Sha^^ JF (2006| Codon oplimizalion of Candida mgosa lipl gene for impro\ing expression in Pichia pastons and biochemical characterization of the purified recombinant LIPI lipase ./ .-liiiu t oml Clicni 54 815-822
.Sauer M, Branduardi P. Ga.sser B. Valli M, Maurer M Porro D, Maiianovich D (2004) DifVcrenlial gene expression in recombinant Pichia pasioris analysed h\
heterologous DN \ microarr;i\ h>bridisaiion Miaoh Celt t-aa3 17,
Dcrveauv S. Vandesompele J. Hellem.ins J (2010) Ho\s Ui do successful gene evpressuin analysis using Real-time PCR Meihods 50, 227-230
Sun "t'. Ll Y. Luo D. Lum DJ (2(112) Pseudoyenes as Weaknesses of AC TB (Acib) and GAl'Dll (Gapdii) Used as Reference Genes m Rc\crse TransLriplion jiid Polj-merase Cham Reaciions Pl.oSOncl 6416"^'*
/.hang W. Zhao HL. Xue C, Xiong XII. Yan \{) Li XY, Cheir HP. Liii /M (2006| Enhaneed seirelion ol heierologous proiems in I'lthia pastons loIlo\Mng overex press I on ol Saechaiomyces eere\isiae chaperone proteins liwicclinol Pi og 22 1090-1095
Zhang W, Inan M \h and opi
MM 121107) R,i[
mon of led-balcli and Meihods Mul Biol 3ii9 4'-h4
lal des
Ye RW. Wang f Bed/\k L, t roker R.\l 121 Apphcanons ot DN \ microarray in niKrobial svsien
\huo MciholA' 257-272 Vandesompele J Dc Prcier N. De Paepe \ Spc
PuppL H, ' . I' I -I'l I'
Le Thi Lan Anh etal geometric averaging of muhiple internal conlrol genes. De Schutter K. Lin YC, Tiels P, Van Hecke A, Glinka S, Genome Biol 3 RESEARCH0034 Weber-Lehmann J, Rouze P. Van de Peer Y, Callewaert N (2009) Genome sequence of the recombinant protein Nolan T, Hands RE, Biislin SA (2006) Quaniification of production host Pichia pastons. \al Biolechnol 27* 561- mRNA using real-time R":f-PCR,.V£//P»r7tor 1 1559-1582 5^6.
V A L I D ATrON O F T R A N S C R I P T I O N OF BLA G E N E E N C O D I N G BETA-LACTAMASE ]\
R E C O M B I N A N T PICHIA PASTORIS S T R A I N S 0 \ E R E X P R E S S I N G I N T E R L E U K I N - 2 A N D A M Y L A S E BY R E A L - T I M E P C R T E C H N I Q U E
Le Thi Lan Anh, Tran Nhat Anh, Than \ an .Minh, Ngu>en Hong Thanh, Duong Cam Thiiy, Le Thi Thu Hong, Truong Nam Hai
Inslilule oJ Biotechnology, Vieinam Acadenn of Science and Technology SUMMARY
Pichia pasioris fP pastons 1 has been used extensively in ilie production of various recombinant proteins of different origins llowever, the molecular mechanisms of recombinant gene expression in this yeast have not been adequately studied Microarray-based transcnptomic analyses have pointed out the differences in transcnpUonal levels of bela-laclamase Ihla/ between conlrol strain and recombinant a-aniylase (Amy) or human lnterleukin-2 (IL-2) strains To clarify Ihe relationship between the expression of bla and rccombinanl Amy/IL-2 genes in P pasioris strains, the transcription levels of those genes need to be evaluated more precisely and detailed with real-time PCR. Real-time PCR technique was used with mRNA samples extracted from recombinant Amy/IL-2 and conlrol strains at 0, 6. 12. 24. 48 h of fermentation after Ihe first methanol induction Transcription levels ofhla in ditTerent samples were compared using Ihe average threshold cycles of three dilTerent fcimentation batches and relabve quantificanon method. The results shown Ihat there were great differences between lecombmanl Amy/lL-2 strains wilh conlrol strains al every time points, but Ihere was no significani difference when comparing recombinant Amy strain with lL-2 strain. The transcription level of bla m recombinant Ainy/IL-2 strains reached maximum value of 45-foId of control strain at time 12 h ume point.
In addition, there were also small differences when comparing expression levels m differeni Ume points. The difference was more than Iwo-folds between different points of the conlrol strain, whereas Ihere was difference belween the 24 h point and 48 h point of ihe IL-2 strain Others compansons had Ihe less-lhan-Iwo value.
Keywords: Ainvlusc. beta-tactuma^e . inicrleiikin-2, Pichiapa,storis. Real-time PCR. Transcriplomics
iiihoi toi correspondence- Tel: ^84-4-37562790; Fax: +S4-4-37562790. E-malh inhada.vnnvn
