Tgp chi Cdng nghe Sinh hoc \\{\y. 69-75, 2013
T H I E T KE V E C T O R A D E N O M R U S ( H A d 5 ) T U T O H O P M A N G G E N C Y T O K I N E ( c h G M C S F ) T A N G C U O N G MIEN D | C H C H O G I A C A M
VQ Thj Tien, Hoang Thi Minh Chau, L l Thi Kim Xuy§n, Le T h a n h Hoa Vi^n Cong nghe sinh hpc, Vien Hdn tdm Khoa hpc vd Cdng nghe Viel Nam
TOM TAT
GM-CSF (granulocyte-macrophage colony stimulating factor) la mot trong nhung loai cytokine duoc te bao CO tham quyen mien dich san xuat truoc Hen \ a tham gia giai doan thir nhat ciia qua Irinh dieu hoa dap img mien dich a ngucn, dong vat, ke ca gia cam GMCSF co kha nang lang cirong dap img mien dich \k de khang b?nh lal hieu qua hem cua co the. He thong vector adenovirus HAd5 (Stralagene) duoc lua chon lam he thong dan truyen gen chGMCSF (chicken GMCSF) de lao adenovirus lai to hop va sir dung dua vao co the de tang cirdng mien djch cho gia cam. Truoc bet, hop gen KZ-chGMCSF duoc kien tao tir san pham PCR (co do dai 462 bp, thu nhan voi cap moi CHF- CHR, voi diem cat BgtW o dau 5" va Xho\ o dau 3') Sau dd hop gen duoc nhan dong vao vector con thoi pShuttle-CMV, tao nen plasmid con thoi lai to hop pShuI CM\ KZ-chGMCSF Vector con thoi Iai to hgp nay duoc dong nhiem Iheo phuang phap Jong nhicm Icc iiep \ao le bao £ coli BJ5183 da mang vector pAdeasy-1 (goi la Ecoli-BJ5183-pAdeas\l) de thirc hien qua Irinh Irao doi cheo va tao ra vector adenovirus Iai lo hop pAd-pShuItle-KZchGMCSF Sang loc p^d pShuule KZchGMCSF bang each nhan dong tren moi tniong chon loc kanamycin, phan cat enzyme gioi han (su dung cai don bang EcoRl:
va cat kep bang BgHl -Xho]) va bang phan img PCR dac hieu \ai cap moi pShutF/pShuiR (bam vao promoter CMV cua pShulUe-CMV) va cap m6i dac hieu gen chGMCSF la CHF/CHR) Ket qua cho thiy. chiing loi da thu nhan duoc DNA plasmid tai to hup mang gen cytokine (chGMCSF) gia cam la pAd-pShuttle- KZchGMCSF, lam nguyen lieu vector kien tao adenovirus liii to hop mang gen cliCiMCSI irong le bao HEK293 sau nay.
Tu'khoa: adenovirus, chGMCSF. ddng nhiem, "liop gen c
DAT VAN DE
Tao adenovirus tai to hap bieu hien va dan truyen gen ngoai lai phuc vu cho san xuat cac che pham the he mm (vaccine, khang nguyen, cytokine. ) doi hoi mpt qua trinh phirc tap bao gom cac cong doan khac nhau tir viec thiet ke plasmid con thoi t^i to hop mang hop gen ngoai lai den viec thu nhan plasmid adenovirus duac chuyen giao hop gen do va cuoi ciing la tao adenovirus tai to hop chiia hop gen ngoai lai de phuc vu yeu cau thuc te iimg dung(LeTlianh Hoa, 2011).
He ihong HAd5 ciia hang Stralagene (AdEasy^^' Adenoviral Vector System), bao gom 2 ioai vector do la vector con thoi pShutlle- CMV. su dung chuyen giao hop gen ngoai tai va vector pAdeasyl chira h^ gen thieu nang ciia adenovirus de tiep nhan va lai 1^0 adenovirus tai to hop trong le bao HEK293 (Le Thanh Hoa, 2011) Ngoai ra. ciing vci cac he thong vector, con co cac dong le bao phu tro; E coli DH5a. £ coll BJ5183. E colt XLIO Gold \a ti bao ill K293. Vector pShuttle-CM\' la mot vong DNA CO kich Ihirac 7.5 kb. trong do. \iing da nhan dong MCS (multiple cloning site) nSm giCra promoter ( M \ va chuoi tin hieu SV40 Vector pShunle-C M \
•• pla.' ml c, d Ivrp
CO kich thuac nho dong vai Iro nhan dong va kien lao hop gen ngoai lai. sau do hop gen ngoai lai se dircTc chuyen vao vector khung adenovirus pAdeasy- 1, nho CO che trao doi cheo gifra hai viing luang dong con goi la cdnh lay irdi sa cdnh tav phai (left arm va nght arm) ton tai tren hai vector nay (Vii Thi Ti^n el ai, 2011. Le Thanh Hoa. 201 1) He thong vector adenovirus HAd5 va plasmid con thoi pShultle-CMV, cung nhu nguyen li kien lao plasmid va vector tai to hgp mang gen khang nguyen da duoc chiing loi mo ta va ap dung doi \cVi gen khang nguyen VP2 cua virus Gumboro gia cam (Le Thanh Hoa el al. 2010. Vu Thi Ti^n ei c;/, 2011. Le Thanh Hoa. 2011) Tuang ly. neu gen cvlokine duoc gai vao he ihong adenovirus, thi adcnoMrus tai to hap mang gen cytokine se san xual loai CNtokine luong img Iham gia dap irng mien dich
Cytokine la cac phan lu h(>a lan. cc) ban chai la glycoprotein hoac protein, do cac le bau co tham quy^n mien dich tiet ra. tham gia hoat diing tin hieu giua cac iC' bao trong dap img mien dich. va duoc phan loai thanh cac interferon, inierlcukin cac yeu i(>
kich Ihich tang smh quan the iCSF. Clonv Slimulating Factor) \a cac ch;U dan tru\cn sinh hoc
Vu Thi Tien ei al (mediator) (Chabalgoity et at, 2007) G\I-CSE
(granulocyte-macrophage colony stimulating factor) la mot trong nhung ioai cytokine dugc san xuat fruac tien, tham gia giai doan thii nhat ciia qua trinh dieu hoa dap img mien dich is ngucn, dong vat, ke ca gia c^m (Chabalgoilyc'/a/, 2007).
Trong tu nhien, a ca the dong vat, hrgng GM- CSF chi dugc san xuat ra vira dii Tuy nhien, khi ca the duoc liem phong vaccine hay bi benh trong tu nhien thi cytokine can duo'c tang cucmg de thyc hien Ifii qua trinh dap ung mien djch GM-CSF duac ma hoa bai mgt to hcip gen tren nhiem sac the, do do, gen GM-CSF c6 the phan lap dugc va chuyen vao cac vector tai U^ hop de bieu hien thanh GM-CSF tai lo hop. trong dieu kien nhan tao (in vitro) hay tren chinh doi tugng dong vat (m vivo) (Chaturvedi el al, 2010), GM-CSF duoc coi la chat bd tro sinh hoc (biological adjuvant), co gia tri tang cuong mien dich, mot khi dugc sii dung trong chinh doi tucmg dong vat (Chabalgoity el al, 2007) Nham chii dgng tan dung vai tro GM-CSF cua gia cam (ki hieu, chGMCSF). chiing toi tien hanh thiet ke vector bieu hien to hap gen chGMCSF bang he thong vector adenovirus HAd5. de tao nen adenovirus tai to hgp mang gen chGMCSF ctia gia cam
Trong bai biio nay chiing loi trinh bay qua trinh ihiel ke vector adenovirus mang gen chGMCSF cua gia cam. kiem tra san pham thiet ke duac tao ra bang cac ky thual sinh hgc phan tu, nham muc dich san xual adenovirus Iai to hop mang gen chGMCSF, sii' dung tang cucmg mien dich cho gia cam.
\ .AT LlEU V,\ PHUONG PHAP Nguyen vat lieu
Hi} lliong vector adenovirus vd plasmid ctm thoi.
do hang Stralagene cung cap bao gom plasmid con thoi pShuiile-CM\'. plasmid adenovirus khung pAdeasy-1; E call chiing BJ5183 cho thuc hien gay dong nhiem, L coli sieu kha bien chung XLIO Gold^ cho san xuat plasmid adenovirus tai to hap; tt bao bac cao dong HEK293 su dung eho lap rap adenovirus lai to hop trong te bao.
Te bcio F toll DH5a (Stralagene) diing de san xuat DNA plasmid con thoi pShultle-CMV va cae thao tac nhan dong khac .
Smh phdm \ d enz\ me gun han Gom cac bg kit i.ich DNA. phisinid tai to hop; kit thoi gel do hang Bioneei (lltin Quoc) cung cap, cac enzyme gioi han
£"coRI. BgiiX, Xhol, Pmel, Pad cua cac hang Fermentas va Neu England Bioiabs,
Ngudn gen chGMCSF. Gen chGMCSE da dugc phan lap va nhan dong trong vector PCR2.I, thyc hien tai phong Mien dich hoc- Vien Cong nghe sinh hoc.
Phuong phap thiet ke hop gen chira chuoi Kozak va gen c h G M C S F
Gen chGMCSF duac phan lap tir ga da duoc nhan dong va luu giir trong vector PCR2 1, Tuy nhien, de gen chGMCSE co the hoat dgng duoc trong he vector adenovirus va te bao chii la HEK293 thi trinh ty ma hoa cua gen, a dau 5', phai dugc nam sau chuoi Kozak va bo ma khai dau va o dau 3 ' , sau gen chGMCSF la bo ma ket thiic Dya vao trinh ttr gen chGMCSE da luu giii va diem cat ciia enzyme gi6i han viing da noi MCS cua vector pShuttle CMV, cap moi CHE - CHR da dugc thiet ke nhu sau:
Mc>i C H E CTC 4 GA rC7lCCACCATGG|ATGCTGGCCCAGC
TCACTATTC 3 ' (phan in nghieng la diem cat cua BglU; phan m dam va dong khung la chuoi Kozak, phan gach ben duai la trinh tu dac hieu gen chGMCSF).
MOi CHR 5'CTTCrCG^GTTAGATGCAGTCTTTCTCCTC
3 ' (phan in nghieng la diem cat cua Xho\; phan gach ben dual la trinh ty dac hieu ciia gen chGMCSF)
Veil cap m6i CHF-CHR, bSng phan iimg PCR, sir dung khuon la DNA plasmid tai to hop luu giir gen chGMCSF (trong pCR2 I), theo tinh toan, chiing toi thu dugc san pham PCR c6 do dai 462 bp, chira loan bg "hop gen cytokine" ki hieu la |BglII-KZ-|
IchCMCSF-Xholl
Phu'ong phap tai thiet ki plasmid con thoi De hop gen dugc chuyen vao diing chieu va diing Vl tri tren vector con thoi pShuttle-CMV, vector nay duoc phan cat bSng BgtW (AGATCT) va Xhol (CTCGAG), ihu doan DNA ciia vector (Sg/il- Xhol) c6 do dai 7,5 kb, bang phuong phap thoi gel (gel-etution) (trinh bay chi tiet, xem Le Thanh Hoa, 2011)
Phuong phap tao t^ bao kha biin tai t6 h ^ E.
coli BJ3183 mang pAdeasy-1
Veclor pAdeasy - I duo'c chuyen nap vao tt bao kha bien E coli BJ5183 bang phuang phap s6c
Tgp chi Cong nghe Smh hpcU{\) 69-75, 2013
nhiet: Chuyen 50 ng DNA vector pAdeasy-l vao 40 pi te bao kha bien E. coh BJ5I83, ii da 45 phiit; s6c nhiet 42°C trong 45 giay, ii da 2 phiit; b6 sung 100 pi moi truong SOC, nuoi te bao trong may lac 200 vong/phiit a 37"C . trai te bao len dTa thach va li 37"C qua dem Cay chuyen khuan lac sang moi truang long CO bo sung khang sinh ampicilm qua dem va tach chiet plasmid pAdeasy-l bang bg kit tach plasmid cita hang Bioneer. San pham plasmid duoc cai mo vong bang enzyme EcoKl va dien di kiem tra tren gel agarose 1%, Ket qua cho san pham DNA co kich thuoc khoang 33,5 kb, dieu do chimg to vector pAdeasy-l da dugc chuyen nap vao te bao E coli BJ5! 83. Te bao tai to hgp nay dugc diing de san xuat thanh te bao kha bien tai to hgp mang vector pAdeasy-l, goi la Ecoli-BJ5l83-pAdeasyl.
Phirong phap dong nhiem tao plasmid tai to hop pAd-pShuttle-KZchGMCSF
De qua trinh Iai U") hop dien ra giCra hai vector pShuttle-CMV mang hop gen Ko7.ak-chGMCSF vm vector khung adenovirus pAdeasy-1 thi hai vector nay phai ton tai irong ciing mpt le bao E, coli chiing IJJ5183 Phuang phap dong nhiem ke tiep (Le Thanh Htia, 201 I) duge sirdyng, mo ta nhu sau. Triroc lien.
DNA vector pAdeasy-1 dugc chuyen nap \ao te bao E. coli BJ3183 bang phuong phap soc nhiet. tiep den, nuoi cay te bao E. coh BJ3183 mang pAdeasy-l de san xuat te bao kha bien tai to hop ggi la Ecoli- BJ5l83-pAdeasy-] nhu da mo ta. sau dd. tiep ujc chuyen nap DNA ciia vector pShuttle-CMV-Kozak- chGMCSF vao Bang each nay. hieu qua dong nhiem cao hern nhieu so vai phucmg phap chuyen nap dong then ea hai vector ciing mgt liic \ a o te bao E coli BJ5183 (Le Thanh HdaT 2011),
KET QUA VA TH.'\0 LL.4N
Thi^t ke hop gen B g l I I - K Z - c h G M C S F - \ h o l va gai vao vector con thoi pShutt!e-CM\'
Nhdn ddng hop gen Bglll-KZ-chGMCSF-Xhol Bang phan img PCR, su dung cap moi CHF - CHR va khuon la gen chGMCSF duac luu giii trong vector PCR2 i. loan bg doan DNA chira diem cat Bglll, chuoi Kozak, gen chGMCSF va diem cat Xhol hop gen Bglll-KZ-chGMCSF-Xhol da duac thu nhan (Hinh 2, trai) va plasmid con thoi pShuttle- C M V tai t6 hgp san pham chiia hop gen Bglll-KZ- chGMCSF-Xhol duac dien di ki^m ira tren gel agarose 1% (Hinh 1)
9,4kb—
6,5kb—
4 , 4 k b -
A]
- - 7 , 5 k b ipShuttle-CMV)
•~8,0kb (pStiultle-CMV-GMCSF|
' ~ 7 , 5 k b (pShutlle-CMV)
' " 4 6 2 b p IKZchGMCSF)
•MM^:
Hmh 1 Di^n di ki4m tra san pham PCR Ihu nhgn hop gen Bglll-KZ-chGMCSF-Xhol. kich thuoc 462 bp tu nguon khuon la cac plasmid pCR2 1 luu gen chGMCSF va plasmid con thoi lai l6 hop mang hop gen Bglll-KZ-chGMCSF-Xhol M marker (DNA Lamda cat bSng Hindlll) Ban di^n di A s6 1 va 2 doan DNA cua hop gen Ban dien di B so 1 pShuttle-CMV cai bang EcoRl (do dai 7,5 kb|, 2 pShullle-CMV-KZchGMCSF cat bang EcoRl (-8 0 kb) 3 pShuttle-CMV-KZchGMCSF cat bang hai enzyme 6g/ll va Xhol (7,5 kb va 462 bp)
De chen hop gen Bglll-KZ-chGMC SI -Xhol \ a o veclor con thoi pShuttle C M \ Ihi can phai phan cat d6ng then ca san pham PCR ciia hop gen Bglll-KZ- chGMCSF-Xhol \ a DN \ eua vector con ihoi bSng
hai enzyme ^,e/Il \ a .V/i<;l. sau do hai s;m pham na^
dugc noi lai \ a i nhau de lao nen \ecior con thoi la lo hpp San pham PCR hc)p gen duirc cat bang ha cn/\-me fii,'/ll \ a Xhol. dien di tren gel agaro'-e I"
V u T h i Tien etal de kiem tra (Hinh IA), sau do cat phan gel agarose
chiia bang DNA co kich thuac 462 bp va dugc tinh sach lai bang kit "thoi" gel (elution) ciia hang Bioneer (Han Quoc). DNA ciia plasmid con thoi pShuttle-CMV (7.5 kb) cung dugc ck bang Bglll va Xhol, dien di phan tach tren gel agarose va tinh sach lai nhu mo ta a tren. Hai san pham tinh sach nay duac dien di kiem tra ,xac dinh ham lugng DNA va thyc hien phan img noi (ligation) vai nhau. San pham noi ghep dugc chuyen nap va nhan dong bang tc bao E. coli DH5a, san pham DNA plasmid tai to hgp duoc tach chiet va cat kiem tra bang enzyme EcoRi va bang hai enzyme BglU va Xho\ (hinh 1B).
Ket qua cr hinh I cho thay bang DNA ciia plasmid tai 16 hap pShuttle-CMV-KZchGIVlCSF khi cat don bang EcoRl, co kich thuac khoang 8,0 kb (Hinh IB, duong chay so 2), so vai doi chung cua vector pShutlle-CMV la 7,5 kb (cit bSng EcoKT).
De3ng thai khi cat kep bang Bglll va Xhol, DNA cua pShuttle-CMV-KZchGMCSF cho 2 bang; 7,5 kb ciia pShuttle-CMV va bang kia 462 bp ciia hop gen BglH-KZchGMCSE-XhoI (Hinh IB, duong chay s6 3), Dieu do cho phep ket luan, DNA ngoai lai la hop gen BgHI-KZchGMCSF-XhoI, da dugc cai vao trong vector pShuttle-CMV. tao nen pShuttle-CMV- KZch GMCSF
Ket qua dong nhiem vector pShuttleCMV-Kozak- chGMCSF vao bao kha biln tai t6 hop Ecoii- BJ5183-pAdeasyl
Nhu vay, vector khung adenovirus pAdeasy-1 luon CO mat trong te bao Eeoli-BJ5183-pAdeasy-l, khi chuyen nap DNA ciia vector pShutfle-CMV- KZchGMCSF da m a vong bang Pmel va loai phosphate a hai dau, thi te bao Ecoli-BJ5l83- pAdeasy-1 se tiep tuc tiep nhan DNA cua vector pShuttle-CMV-KZchGMCSF va c6 kha nang mang DNA ciia ca hai loai vector (Le Thanh Hoa, 2011, Vu Thi Tien el ai. 2011), Nhu vay, ve nguyen tSc, qua trinh tai to hgp kep se dien ra trong Ecoii- BJ5183-pAdeasy-], de tao ra DNA cua plasmid vector adenovirus tai to hgp mang hop gen KZchCMCSF. Co 2 truang hgp xay ra trong t^ hao dOng nhiem Ecoli-BJ5183-pAdeasy-l" i) Se chi lon tai vector khung pAdeasy-1; li) Vector tai to hop giOa pAdeasy-1 vai pShuttle-CMV-KZchGMCSF dugc dat ten la pAd-pShuttle-KZchGMCSF, D^
thu dugc vector tai to hgp, chung toi phai tien hanh sang Igc san pham bang khang sinh kanamycin, Truac tien, khuan lac dugc nuoi cay tren moi tru'dng long CO bo sung kanamycin va tach chiet DNA plasmid tir te bao nuoi cay va dien di kiem tra tren
% (Hinh 2).
Hinh 2, Ket qua tach chiel plasmid tai lo hop pAd-pShuttle-KZchGMCSF kiem Ira tren thach agarose 1% Ghi chu. M Marker Lamda cat bing H/ndlll, Ou-ongchay 1 - 8 DNA plasmid tach lir cac clone sang loc so 1 d i n 8 (mul ten chi din]
Ket qua dien di a hinh 2 cho Ihay, plasmid tai to hcyp CCI kich thutre eao hon 23 kb ciia marker, thyc chat khoang 34 kb, cho biel kha nang qua trinh tai to liop da dien ra. Do san pham tai to hgp (pAd- pSluililc-KZchGMCSF) va veclor adenovirus khung (p.A.deasyl) co kich thuac gan bang nhau nen rai kho cci ihe nhan biet hai san pham nay bang phuang phap dien di tren thach agarose 1% (Hinh i. mui ten chi dan), Thay vao dc). do veclor adenovirus khung co
chira gen ampicillin va vector adenovirus tai to hgp CO chira gen kanamycin, nen phuang phap sang loc bang khang sinh duge sir dung de tach dong phan biet hai loai vector nay.
Sang ioc thu nhan vector tai to hgp pAd-pShuttle- KZchGMCSF
Vector pAdeasy-l mang gen khang khang sinh ampicilin, Irong khi do vector Iai to hgp pAd-
Tap chi Cdng nghe Sinh hpc n(l): 69-75,2013 pShuttle-KZchGMCSF chi mang gen khang khang sinh kanamycin (gen khang sinh ampicilin co tren vector pAdeasy-l trucfc khi tai to hgp, da bi loai bo trong qua trinh dong nhiem). Plasmid tai to hgp lach tir clone I den clone 8 thu dugc 6 tren, dugc chuyen nap vao te bao E. coli sieu kha bien chung XLIO Gold va nuoi cay tren moi truang sang loc chi bo sung kanamycin, Te bao vi khuan tiep nhan vector pAdcasy-1 sc khong sinh truang dugc tren moi truang co kanamycin, ma chi co te bao vi khuan mang vector tai to hap pAd-pShuttle- KZchGMCSF phat trien va hinh thanh khudn lac
23kb_
9,4kb -
Tuong irng, tren moi dTa tc bao chiing loi chgn 2 khuan lac de nuoi tren moi truong long co bo sung kanamycin, tach chiet DNA plasmid va dien di kiem tra san pham, Ket qua thu dugc trinh bay a hinh 3
KCI qua a hinh 3 cho thay. hau het cac clone deu cho DNA plasmid co chat lugng toi, ki'ch thucrc cao hem vach DNA cao nhat ciia chi thi DNA Lamda (Hinh 3), Nhung khuan lac nay la img vien chira plasmid lai to hgp pAd-pShutlle-KZchGMCSF can thu nhan.
Hinh 3: Dign di sang lpc plasmid tai to hp'p pAd-pShullle-KZchGMCSF, gom DNA tach chiel l u 14 khu;
Ket qua kiem tra san p h a m pAd-pShuttle- KZchGMCSF bSng enzyme gicri han
San phdm dicn di cho Ihay DNA plasmid thu duoc CO kich ihudc kha IcJn, De khang dinh, cac clone nay co diing la chiia cac vector pAd-pShuttlc- KZchCiMCSF, cdn li^n hanh kiem Ira bang enzyme gicri han va b3ng PCR dac hicu, Theo nguyen li, Iren vector con thoi pShutdc-CMV va veclor khung adenovirus pAdeasyl dau duac thict ke mot vi tri cat ciia enzyme gicJi han Pad, ma khi hai vector nay Iai l6 hgp tuong dfing vai nhau tai cac viing cdnh lay Irai va cdnh lay phdi sc cho ra vector adenovims lai l6 hgp mang hai di6m cdt ciia enzyme Pad (xem Lc Thanh Hoa, 2011),
Do do, chung toi ldy hai mau la DNA ciia pAd- pShutilc-KZchGMCSE cua c l l / l va cl4/l trinh bdy 6 hinh 3, cdt bdng Pad, dong thoi DNA ciia vector pAdcasy-1 cung dugc cat de lam doi chiing, Dien di san phdm phan ciit Ircn gel agarose 1 % cho ket qua trinh bay a hinh 4 K5l qua cho thay Pad da cat san phdm vector tai to hgp cho ra hai bang DNA, ki'ch ihiroc bang lon !a - 30 kb va bang nho la 4,5 kb, dung nhu dy linh (Hinh 4)
Hinh 4 Anh dien di kiem Ira ket qua phan c^l pAd-pShutlle -KZchGMCSF clone 1/1 va clone 4/1 biing enzyme P a d , Hai bang DNA (-30 kb va 4,5 kb) dupc nhin Ih^y, Irong khi DNA plasmid pAdeasy-1 chi cho bang co kich thucrc khoang 33 kb
Theo qui trinh sang lgc plasmid adenovirus tai to hgp (xem He, 2004, AdEasy^"" Adenoviral Vector System), khi phan cdt vector tai lo hgp bdng Pad se cho hai doan DNA, bao gfim mot doan co kich thucrc 73
VQ Thi Tien et ai lon khoang 30 kb va mgl doan co kich thuac nho,
hoac la 3,0 kb (nlu qua trinh tai to hgp dien ra tai
\-iing cdc cdnh lay trdi) hoac la 4,5 kb {neu qua trinh tai to hop dien ra tai cac vi tri khdi ddu sao chep) (He, 2004) Cac clone chiia cac san pham plasmid nhu the nay nen duac kiem tra sang Igc va nhu vay, clone 1/1 va 4/1 ma chung toi .sang loc cho ket qua diing la cac clone tai to hpp chiia vector pAd- pShullle-KZchGMCSF.
Kiem tra san pham pAd-pShutfle-KZchGMCSF bang phan iing PCR
Dc chac chan hon nua ve san pham plasmid tai 16 hap thu dugc la p A d - p S h u t t l e - K Z c h G M C S F , chiing toi diing hai cap moi de kiem tra bang phan irng PCR, g6m cap m6i PSHUTF-PSHUTR bam tren Irinh nr vector pShuttle-CMV, neu co mat cua hop gen KZchGMCSF thi san pham PCR co ki'ch thudc khoang 0,5 kb; va cap m6i SHUTF-ADER (mfii
SHUTE bam tren trinh ty pShuttle-CMV, con ADER bam tren trinh ty ciia pAdeasy-1), neu qua trinh tai to hgp dien ra thi se cho san pham PCR cd kich thucrc - 2,6 kbj. Chiing toi lay ngau nhien 3 mau la DNA ciia cac clone 1/1; 2/1 va 4/1 lam khuon, va vector pAdEasy-l lam khuon doi chiing am, de ihuc hien phan ling PCR. San pham duac dien di tren gel agarose, trinh bay a hinh 5 Ket qua cho thay, san phdm PCR bdng cap moi PSHUTE- PSHUTR co kich thuoc khoang 0,5 kb va san pham PCR bang cap moi SHUTF- ADER co kich thuoc khoang 2,6 kb, diing nhu dir iinh. Irong khi do doi chiing am voi khuon la pAdeasy-l, khong cho san pham PCR do khong CO Vl tri bam cho cac cap moi thuc hien phan ling,
Nhu vay, cac ket qua kiem tra bang enzyme gioi han va PCR deu cho thay, san pham DNA taeh chiet tir cac clone l / l , 2/1 va 4/1 deu chiia plasmid adcnovini.s tai to hgp pAd-pShuttle-KZchGMCSF,
IVI6i cho phan i i n g PCR
Hinh 5, Kel qua kiem Ira su' co mat ciia hop gen KZchGMCSF b^ng cap moi PSHUTF- PSHUTR (co kich Ihu'O'G khoang 0,5 kb) va va qua trinh tai to hop bang c$p moi SHUTF- ADER (san pham khoang 2,6 kb).
Da Ihiet ke thanh cong hop gen KZchGMCSF co day dti cac thanh phan can thiet cho su bleu hien ciia gen liong te bao chii HEK293, va cai thanh cong vao plasmid con thoi pShutlle-CMV. Bang phuong phap dong nhiem kc tiep trong E coli chiing BJ5183, vector adenovirus tai to hgp mang hop gen KZchGMCSF (pAd-pShiittle-KZchGMCSF) da duac thu nhan, sang lgc va kiem tra xac dinh bang enzyme giai han va PCR, va sc dugc dimg de tao adenovirus tai to hgp mang gen cytokine chGMCSF tren tc bao HEK293 sau nay.
Len cam o'n: CcJng trinh duac llnrc bien vdi su tin Ira kmh phi de ldi nhanh do PGS. TS Le Tlianh Hoa chii nhiem, cda Chuong irinh CNSH ndng nghiep (chu nhiem de ldi chinh TS Le Thi Hdng Minh) vii Phong Thi nghieni trgng diem cdng nghe gen, Vien Cdng nghe sinh hgc.
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Genetics John Wiley& Sons, Inc ^ '^^^"^ "^^ '20! 1). TTiiet ke plasmid con thoi pShuttle- CMV mang "hop gen" VP2 ciia virus Gumboro de tao Le Thanh Hda (2011) Sach chuyen khao: '"CcJug nghe veclor adenovirus tai 16 hop Tap chi Cdng nghe Smh lioc, adenovirus vd nguyen ly tao veclor tdi to hop". Nhd 8(3), 281 -289,
( (INSTRUCTION OF A RECOMBINANT HAd5 ADENOMRUS XTCTOR HABORING CYTOKINE GENE (chGMCSF) FOR ENHANCEMENT OF IMMUNITY IN POULTRY
Vu Thi Tien, Hoang Thi IVlinh Chau, Le Thi Kim Xuyen, Le Thanh Hoa' Inslilule of Biolechnotogy, Vietnam Academy of Science and Technology
SUMMARY
GM-CSF (granulocyle-macrophage colony stimulating factor) is one of cytokines produced b mmu elTecIor cells in the earliest stage, participating in the regulation of the immune response in hun an n n 1 including chickens. GMCSF, if administered as a biological adjuvant, will enhance the body lo egula e I e immunosyslems working well against infectious diseases Adenovirus HAd5 system (Stralagene) hosen for construction of a recombinant adenovirus veclor habormg the cylokme gene chGMCSF. for enhancemcni of immunity m pouliry. First of all. Ihe cytokine casscUe KZ-ehGMCSF was generated from the PCR product (size of 462 bp. ainpliOed by CHF- CHR speeillc primerpairs). Tins cytokine casseUe then was donned into the pShuUlc-CMV plasmid to construct a rccombinanl veclor. namely, pShut-C\I\-KZ-ch(;MCSF This veclor was Iransl'eclcd by the method "suhseipieni homologous icumihinaiion". into E (oli BJ5183 cells previously haboring the pAdeasy-1 veclor (namely. Ecoli-B.I518.VpAdeasyl). in order to facihtaie homologous reconibmalion of these iwo DNAs for generation of rccombinanl adenoviral plasmid. as pAd- pShuttlc-KZchCMCSF Screening this rccombinanl plasmid wiis earned out by culturing reconibiiianl E coli cells m a kanamycine-containing mednim Selection of positive rccombinanl plasmid by rcsirielion enzyme digestions (single digeslion by EcoRl, and double by Bgt\l-Xhol) and specific PCR using PSllUTF/PSHUTR primerpairs (both binding on CMV promoter sequence, yielding a produci of 2 6 kb) and cliGMCSF-specific CHF/CHR primers (internal primers of chGMCSF. resulung in a produci of 462 bp). Rcsulls indicated thai the adenoviral DNA plasmid containing the chicken GMCSF gene (ch(rMl_SF) was obtained, and that was confirmed as pAd-pShuttle-KZchGMCSF Successlhl construction hereby described, pro\ides \eclor materials for subscquenl conslruction of llic chGMCSF-recombinani adenovirus in ihe MFK2y.l cells in the next stage
heywonls, adenovirus, chGMCSF. ••cylolniw cas.scllc", homologous laombinaium. ictomtwianl plasmid
' Author for correspondence E-mail imiblvniu a/naiicom
