Biểu hiện và tinh sạch hai protein vỏ CP và CPm tái tổ hợp của virus CTV trong vi khuẩn E.coli

Tap chi Cong nghe Sinh hoc 8(3): 297-302, 2010

BIEU HIEN VA TINH SACH HAI PROTEIN VO CP VA CPm TAI TO HOP CUA VIRUS

CTV TRONG VI KHUAN E. COLI

Dang Th| Hu-ong, Le Van Sffn, Le Trln Binh, Chu Hoang Ha

Vien Cong nghe sinh hoc

TOM TA T

Citrus Tristeza virus (CTV) la nguyen nhan gay benh tan lui (Tristeza) d cac loai cay an qua co mui.

Protein v6 ciia vius (CP va CPm) c6 chirc nang bao boc nucleic acid tranh su phan huy cua enzyme nuclease,

giu: cho hinh thai va kich thudc virus on dinh, dong thdi tham gia vao su hap thu cua virus len vi tri dac hieu

tren te bao man cam. Protein v6 mang tinh kliang nguyen dac hieu nen thudng dugc chgn lam nguyen lieu

trong viec tao cay chuyen gen co kha nang khang dugc virus gay benh d thuc vat va de san xuat khang the tir

de xac djnh chinh xac virus gay benh d cay trong cQng nhu kiem tra mire dp nhiem benh cua cay trdng. Kiem

soat giong sach benh la mot trong nhung bien phap quan trong doi vdi viec san xuat giong cay an qua co miii.

Tir nguon nguyen lieu ban dau la gen ma h6a CTV-CP va CTV-CPm phan lap d Viet Nam, chung toi da thiet

ke vao vector bieu hien pET21a(+) va bien nap vao chung E.coli Rosetta-gami^". Dudi dieu kien cam img 1

mM IPTG d 22°C trong 16 gid CTV-CP va CTV-CPm da dugc bieu hien co kich thudc tuang img khoang 25

kDa va 27 kDa. Tren co sd xac dinh CTV-CP va CTV-CPm trong phan dich tan cua te bao, chiing toi da tinh

sach dugc khang nguyen nay vdi mot lugng Idn, du tieu chuan gay mien dich tao khang the. Day la ca sd cho '

cong tac phuc vu phan tich nhanh tac nhan gay benh ciing nhu danh gia muc dp sach benh cua cay trong.

Til khoa: Vector bieu hien. Virus vdng lui (CTV), protein vd (CP), tinh sach protein

MODAU

Citrus Tristeza virus (CTV) la nguyen nhan gay

benh tan lui (Tristeza) d cac loai cay an qua cd mui,

lam thiet hai nghiem trgng cho nganh ndng nghiep

nhieu qudc gia tren the gidi (Vives et al, 1999;

Aylldn et al, 1999). Bieu hien cua cay bj benh la:

phien la cong hinh thia, d mat gd nai tiep giap gdc

ghep va than ghep cd cac vet Idm hinh thoi nhd li ti.

Klii cay bj benh cho ra rat nhieu hoa va qua nhung

chi vai nam cay tan lui va chet (Tran The Tuc, 1997).

Dua vao trieu chung ciia benh, ngudi ta chia CTV

thanh 5 chiing chinh: seedling yello'ws virus,

grapefruit stem pitting virus, grapefruit stunt bush

virus, lime die-back virus, Ellendale mandarin

decline virus (Price, 1970).

CTV thudc chi Closterovirus, hg

Closteroviridae (Sapanari et al, 2008). Virion ciia

CTV cd dang sgi xoan kich thudc khoang 2000 x 12

nm. Genome cua CTV la sgi don duong RNA cd

kich thudc khoang 19,3 kb bao gdm 12 khung dgc

md (ORE) va 2 viing khdng ma hda (UTR) dlu 5' va

dlu 3 \ ma hda cho it nhat 19 protein bao gdm 2

emzyme protease tuong tu papain, cac protein lien

quan tdi su tai ban (RNA polymerase, helicase,

methyltransferase), protein gidng vdi heat shock

protein HSP70, protein p23 tuong tac vdi RNA,

protein p20 dugc tich luy trong the vui va ma hda

cho mdt sd protein chua rd chirc nang nhu p6I, pI3,

pi8 (Saponari et al, 2008; Rubio et al, 2001;

Satyanarayana et al, 1999). Vd protein bao gdm 2

protein CP va CPm cd kich thudc phan tir tuong irng

khoang 25 kDa (chilm 95%) va 27 kDa (chilm 5%o)

(Yang et al, 1997). Protein CP cau tao nen thanh

phan vd chii yeu (major coat protein) cua virus cdn

proten CPm cau tao nen thanh phan vd thir yeu

(minor coat protein) tao thanh cau tnic kieu "ran dudi

chudng" d dau 5' cua virion. Cau tnic virion bat

thudng nay lan dau tien dugc phat hien trong BYV

(Gago-Zachertefa/., 1999).

Hien nay, d nude ta benh Tristeza dugc coi la

mdt trong cac benh nguy hiem anh hudng tdi nang

suat va pham chat cay an qua cd mui. Kiem soat

gidng sach benh la mdt trong nhung bien phap quan

trgng ddi vdi san xuat gidng cay an qua cd miii. Viec

phat hien benh sdm cd y nghia to Idn trong cdng tac

phdng trii', kiem soat dugc ngudn gay benh va giam

thieu that thoat trong thu hoach. Vdi mong mudn

giam chi phi cha cac phat hien virus Citrus Tristeza

tren cay an qua cd miii, dan thay the cac bd Kit nhap

tir nude ngoai bang chinh nhirng bd Kit dugc san

xuat trong nude. Trong bai bao trudc, chiing tdi cdng

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