Thiết kế plasmid con thoi pShuttle-CMV mang "hộp gen VP2" của vius gumboro để tạo vector adenovirus tái tổ hợp

Tap chi Cong nghe Sinh hoc 8(3): 281 -289, 2010

THIET KE PLASMID CON THOI pSHUTTLE-CMV MANG ''HOP GEN VP2" CUA

VIRUS GUMBORO DE TAO VECTOR ADENOVIRUS TAI TO HOP

Vu Thi Tiln, Hoang Th| Minh Chau, Le Thj Kim Xuyin, Le Thanh Hoa

Vien Cong nghe sinh hoc

TOM TA T

Trong chien luoc tao adenovirus tai to hgp mang cac gen ngoai lai, can co mot plasmid con thoi (shuttle

plasmid) co vai tro trong qua tinh trao doi cheo chuyen gen vai DNA cua adenovirus. Plasmid con thoi

pShuttle-CMV (Stratagene Inc.), sir dung trong nghien cim nay, la mot vong DNA kich thudc 7,5 kb, co mot so

diem cat ciia enzyme ban che de tiep nhan ''hop gen khdng nguyen" cai vao, tao nen vong DNA tai to hop. Sau

do, DNA plasmid con thoi duoc dong nhiem (trao doi cheo thanh phan gen) vai DNA ciia plasmid adenovirus

khung (HAd5) trong te bao E. coli thich ung (dong BJ5183), de tao ra DNA adenovirus vector tai to hp'p mdi.

Til' do, phan DNA cat bang Pac\ se duoc lay nhiem te bao HEK293 de tao adenovirus mai. Trong cong trinh

nay chiing toi thiet ke plasmid con thai mang "hop gen VP2" (VP2 - antigenic cassette). Gen VP2 dugc thu

nhan bang phan irng PCR vdi cap moi GKPKZF-GNOR, tach dong va kien tao "hop gen VP2" co day du cac

thanh phan co ca chuoi Kozak va cac diem cat Kpnl va Notl. Doan DNA hop gen VP2 dugc dugc xu ly bang

enzyme Kpnl va Notl va gai vao DNA plasmid pShuttle-CMV cung cat bang Kpnl va Notl. Sau khi chpn loc,

plasmid con thoi tai to hgp (ky hieu: pShuttle-CMV-VP2), kich thuoc ~ 8,9 kb dupe kiem tra bang enzyme cat

gidi han, bang PCR dac hieu gen VP2 va bang giai trinh tu xac dinh dau 5' va 3' ciia hop gen. Ket qua da thu

dugc mot so pShuttle-CMV chua hop gen VP2 de chuan bi cho kien tao plasmid adenovirus tai to hgp sau nay.

Tie khoa: adenovirus, dong nhiem, "hop gen khdng nguyen ", plasmid con thoi, tdi to hop

DAT VAN DE

Adenovirus trong tu nhien dugc chia lam 4

nhdm chinh nam trong hg Adenoviridae la

Mastadenovirus, Aviadenovirus, Atadenovirus va

Siadenovirus (Bunchen-Osmond, 2003). He gen

adenovirus ciia dgng vat bac cao cd kich thudc

35.800 - 36.200, ciia chim khoang 42.000 - 43.000

nucleotide. Ddi vdi muc dich thiet ke vector, laai

adenovirus thich hgp da dugc cat bd viing gen khdng

can thiet, tai dd gen khang nguyen dugc dua vao de

thiet ke he gen hoat ddng (Impler, 1995; Curiel,

Douglas, 2002). He thdng vector adenovirus dan

truyen gen khang nguyen cd dac tinh thich ung te

bao da vat chu, vd hai vdi nhieu laai, nhan len manh

khi nudi cay tren te bao thich irng {in vitro) de san

xuat virus lam vaccine, cd kha nang dn djnh nhan len

sau khi dua vaa ddi tugng hudng vaccine {in vivo) de

duy tri khang nguyen va san sinh dap iing mien djch

djch the, trung gian te bao va mien djch niem mac

(Bangari, Mittal, 2006; Le Thanh Hda, 2006; 2009;

Parke/a/., 2009).

He thdng HAd-5 (Human adenovirus serotype 5)

ngudn gdc tir adenovirus ngudi, dugc chgn lam

vector bilu hien va dan truyen gen khang nguyen

huu hieu cho vaccine the he mdi gay miln djch, cho

lieu phap gen chdng ung thu va nhieu irng dung khac

(Barauch, Nabel, 2005; Park et al, 2009). Trong

chien luge tag adenovirus tai td hgp mang cac gen

ngoai lai dj loai (gen khang nguyen, gen cytokine,

RNA can thiep), can cd mdt vector quan trgng, cd

vai trd trong qua tinh trao ddi cheo chuyen gen vdi

DNA cua adenovirus, dd la plasmid con thoi (shuttle

plasmid). Plasmid con thoi la mdt DNA mach vdng

kich thudc vira (dudi 10 kb), cd mdt sd diem cat cua

enzyme ban che thich hgp de tiep nhan "hop gen

khdng nguyen" c&x v&o, tao nen vdng DNA plasmid

con thai tai td hgp; va sau dd, cd kha nang dong

nhiem (trao ddi cheo thanh phan gen) trong te bao E.

coli thich irng (ddng BJ5183) vdi DNA cua plasmid

adenovirus khung de tao ra DNA adenovirus vector

tai td hgp mdi (Le Thanh Hda, 2009).

Gin day, dl tai KC04.24/06-10 da dugc nha

nude cap kinh phi de irng dung he thdng adenovirus

lam md hinh vector mang gen VP2 ciia virus

Gumboro tao vaccine cho gia cam. Mdt phan trong

tien trinh thiet ke vector adenovirus la can cd

plasmid chu'a '^hop gen" day dii ciia gen khang

nguyen VP2. He thdng adenovirus vector (ngudn gdc

tir HAd5) ma chung tdi sir dung cd ten ggi la

pAdEasy-1, la vdng DNA khep kin chira he gen cua

adenovirus HAd-5 ciia ngudi, do hang Stratagene

281