Tách dòng và biểu hiện gen Enterocin P ở dạng dung hợp với CBD-SSP Intein trong Escherichia Coli Er2566

Tgp chi Cdng nghi Sinh hgc 1(1): 17-33, 2009

TAC H DON G VA BIEU HIEN GEN ENTEROCI N P d DANG DUNG HQfP VQfl CBD-SSP

INTEIN TRONG ESCHERICHIA COLI ER2566

Nguyin Thanh Nhan', Le Thu Ngoc^ Do Thi Huyen', Tran Ngoc Tan', Pham Thiiy Linh', Le Van

Tru-dmg', Truonng Nam Hai'

Viin Cdng nghi sinh hgc, Viin Khoa hgc vd Cdng nghi Viet Nam

Trumg Dgi hgc Khoa hgc Ttr nhiin, Dgi hgc Qudc gia Hd Ngi

Viin Hda hgc, Viin Khoa hgc vd Cdng nghi Viet Nam

TOM TAT

Enterocin P (EntP) la mot bacteriocin thupc ldp Ila duac tong hap ttr chung vi khuan Enterococcus

faecium P13, c6 hoat tinh lie che sir sinh truang cua cac vi khuan gay benh Gram duang thudng dugc phat hien

ttong nong san thuc pham nhu Listeria monocytogenes, Staphylococcus aureus. Viec tong hap EntP tai to hap

da duac nghien cim ttong he bieu bien Escherichia coli ER2566. Doan gen entP dai 132 bp ma hoa cho EntP

hoan chinh duac dua vao vector bieu hien pTWIN dudi su dieu khien cua promoter T7lacl de tao thanh

plasmid pTWIN-entP. Khi duac cam ung bdi IPTG, chimg E. coli mang vector tai to hap da tong hap doan

protein dung hpp giiia Ssp intein va EntP. Viec bieu hien d dang dung hop nay co the giup cho doan peptide

ngan nhu EntP (khoang 4,4 kDa) tranh bi phan cat bai protease cua vat chu. Sir co mat cua doan cau tnic gan

vdi chitin (chitin binding domain, CBD) ttong Ssp intein giup chung toi phat hien dugc protein lai tai to hop

bang each sir dung khang the dac hieu cho CBD. Dong thdi, do co ai luc cao vdi hat chitin nen protein ngoai lai

dugc giii lai tten cpt sac ki ai luc vdi gia the la chitin, va vdi viec thay doi pH ttong dung dich dem thu mau,

doan EntP de dang dugc cat khdi dang dung hgp. Ket qua thir boat tinh khang khuan cho thay EntP sau khi

dugc tinh sach c6 kha nang ire che su sinh trudng va phat trien ciia S. aureus. Viec bieu hien va tinh sach thanh

cong EntP tai to hgp d dang cd boat tinh sinh hpc ttong te bao E. coli co y nghTa quan ttgng doi vai viec nghien

ciiu san xuat EntP tai to hgp nham iing dung ttong cong nghe bao quan nong san thuc pham.

Tie khoa: entP, Escherichia coli ER2566, Intein I, pTWINl, S. aureus

MO DAU

Bacteriocin la nhiing peptide dupc vi khuan tdng

hpp ra de lie che sy sinh trudng ciia cac vi khuan

khac cimg chung sdng vdd nd. Trong nhihig nam gan

day, cac bacteriocin dupc phan lap tit vi khuan sinh

lactic acid (lactic acid bacteria, LBA) da thu biit

dupc sy quan tam ciia cac nha khoa hpc ttong viec

nghien cuu de su dung trong bao quan thye pham

(Cleveland et al, 2001). Uu diem noi bat ciia cac

bacteriocin la an toan cho ngudi va dpng vat (Galvez

et al, 2007). Hien nay, da cd hai bacteriocin dupc

san xuat tren thi tradmg la nisin, dupc Cue quan ly

thye pham va dupc pham Hoa Ky chimg nhan vdi

ten thuang pham la Nisaplin^"^ (Danisco,

Copenhagen, Dan Mach), va pediocin PA-1 vdd ten

thucmg pham la ALTA™^ 2431 (Kerry Bioscience,

Carrigaline, Co. Cork, Ai len) (Settanni et al, 2008).

Hau het cac bacteriocin la nhiing doan peptide

tich dien duomg, dai 30 - 60 amino acid va dupc

phan tlianh 4 ldp (Klaenhammer TR, 1993). EntP la

bacteriocin lap II sinh ra tit E. faecium P13 va mot

so chiing khac thudc chi Enterococcus. Khi dupc

tdng hop a ribosome, bacteriocin nay la mpt doan

tien peptide gdm 71 amino acid. Hai muai bay

amino acid dau ma hda cho peptide tin hieu cdn 44

amino acid tiep theo ma hda cho peptide trudng

thanh. Peptide nay dupc tiet ra ngoai bao de tieu diet

hoac uc che vi khuan dich thong qua viec hinh thanh

Id tten mang sinh chat cua vi khuan. Tit cac 16 mang

nay, ion K"^ bi giai phdng ra ngoai lam mat can bang

ion giiia mdi tradmg ttong va ngoai te bao (Herranz

et al, 2001). EntP la peptide chiu nhiet cao (60 phut

d 100°C hoac 15 phiit d 12rC) va cd kha nang boat

dpng trong khoang pH rpng tit 2 den 11. Hoat tinh

ciia EntP dn dinh trong thdi gian dai khi bao quan d

4°C va -20°C. EntP cd phd tac dyng tuomg ddi rpng

ddi vai cac vi khuan Gram duomg gay benh thudng

dupc phat hien ttong thye pham nhu L.

monocytogenes, C. perfringens, C. botulinum, S.

aureus (Cintas et al, 1997). Do do, EntP cd tiem

nang iing dung ttong bao quan ndng san thye phim.

Tuy nhien, chiing sinh EntP thudng tilt ra doc tl. Vi

vay, peptide nay da dupc cac nha khoa hpc nghien

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