Tap chi Cong nghe Sinh hpc 13(2): 379-385, 2015 TAO DoNG BIEU HIEN NISIN TREN BE MAT TE BAO NAM MEN SACCHAROMYCES CEREVISIAE BANG DUNG HOP VefI GEN MA HOA a-AGGLUTININ Nguyen Hien Nghia, Thai Ngoc Anh, Nguyen Quynh Hwang, Ng8 Thi CAm Nhung, Nguyen Tri Nhan, TrAn Linh Thtrac, D#ng Thi Phtrong Thao Truerng Dgi hpc Khoa hpc to nhien, Dgi hpc Quac gia Theinh pha Ho Chi Minh Ngay nhiin bai: 19.5.2015 Ngay nhfin ding: 30.6.2015 TOM TAT Nisin duqc biet den nhu bacteriocin ci ph6 khang khuan remg va irc the cac vi khan gay benh tren ngueri va ging vet nhu Staphylococcus aureus, Listeria sp Ben canh Q6, mic di Nisin thu nhttn tir djch len men ching Lactococcus lactis ttr nhien a duqc ling dung remg rii c8ng nghiep bao quan thuc phim, nhien cac chi ng to nhien bieu hien Nisin veri ham !yang thap, di hien nghien cuu va ngosi mac Escherechia colt va nam men Saccharomyces cerevisiae (S huang tai vies san xat Nisin tai to hqp cerevisiae) NM= tim met cach tiep cent mdri di thu nhCin va ling dung bacteriocin brio quitn thuc phim cling nhu tic the vi khuin gay benh chin nu8i gia sic va nu8i thug hai san Trong bai bao nay, chin toi trinh bay chilli ltrqc bleu hien Nisin tren be mit to bao am men S cerevisiae bang phuong phip dung hqp gene nisin v6i gene a-agglutinin Deng to bac) nem men se duqc thu nhan va huding tdri lam nguyen lieu cho nghien cuu ling dung bao quan thuc phitm ding nhu cac the phim irc the vi sinh vitt gaY bOnh• Tir khda: Agglutinin, bacteriocin, cong ngh m(sit, hui,nh (pang nisin pICAS-1, Saccharomyces cerevisiae MO DAU kiem soat tic titian gay benh cna vi sinh v#t tucmg lai (Yang et al., 2014) Bacteriocin la nheng peptide hay protein khang khan, vi khan nham Grain am hoc Gram duong sinh Bacteriocin cilia vi khan Gram ducmg, d#t biet la cilia nhom vi khuan lactic hay goi la nhom vi IchuAn LAB (lactic acid bacteria) duqc nghien tiro va irng dung King nhieu linh Arc nhu c8ng nghiep bao quan thuc phim, y ductc vA probiotic (Chen, Hoover 2003) Nisin la bacteriocin thuec nhom lantibiotic, duqc sinh tong hop bfri Lactococcus lactis (L lactis), peptide truang china 34 amino acid, khei ltrqng pha'n tin 3.3kDa, Nisin to nhien c6 kha nang khang vi khuin lactic, Bacillus sp, Listeria sp va Micrococcus sp (Zendo et al., 2003; Fukao et al., 2008) Ben canh de, nhieu thin nghiem in vitro cho thAy lantibiotic c6 khA Wing khang cac vi khan gay berth ten nguai va deng vet bao gem ca cac vi khuAn khang khang sinh nhu Staphylococcus aureus khang methicillin (MRSA), Enterococcus faecalis khang vancomycin (VRE) (Piper et al., 2009) Ca the tic deng mkt bacteriocin 'chic vei thuoc khang sinh th8ng thuang, c6 xu the di{C tren cac loci vi khan gay benh, nen ching co the coi nhu met ngu6n mei, dAy flan' Wing cho skr Vi khan lactic ke ca sin phAm ctia chimg nhu bacteriocin duqc ding nhtln an toil' va duqc 1mg dung nhieu c8ng nghiep thuc phim Tuy nhien, sir bleu hien him Wong thip la nhtrqc diem dm cac ching sinh bacteriocin to nhien, de nhieu nghien dm dA huang den bieu hien bacteriocin tai to hqp cac he thong to bao chit khic nhu Escherichia colt (E colt), Saccharomyces cerevisiae (S cerevisiae), LAB (Chen et al., 2012; Mierau, Kleerebezem, 2005; Reenen et al., 2003) Nim men S cerevisiae duqc ang **mg nhieu cong nghiep thuc phAm va duce cong nh#n la an town GRAS (generally regarded as safe), nhieu nghien ciru ve san xuet bacteriocin tai to hqp ta S cerevisiae dA c8ng nhu Pediocin (Schoeman et al., 1999), Enterocin L50 (Basanta et al., 2009) Song song d6, nhfmg thAnh cong ye cling nghe be met nem men dA ma met huang phat trien mei cho ding nghe protein tai to hqp Cong nghe be met to bao nem men la phuang phap bieu hien protein muc lieu bing dung hop gene nhAm dung hqp protein muc tieu yeti protein tren ming to bao nem men nhu FLO1, Cwps va a-/a-Agglutinin (Ueda, Tanaka, 2000) Trong bai 379 11 Nguyen Hieu Nghia et al bao nay, chilli* toi trinh bay cac ket qua thief ke too clang to bao nam men S cerevisiae mang dung hop gene bacteriocin vbi a-Agglutinin va bieu hien Nisin tren be met to bao nam men VAT LIEU VA PHtJONG PHAP Chung Escherichia coli DH5a [F, 0801acZAM15, recAl, endAl, hsdR17 (rk, Ink+), phoA, supE44, thi-1, gyrA96, relA 1] (Takara, Nhet) dung lam brio chic de nhan ban plasmid Chung Saccharomyces cerevisiae W303 [MATaIMATa {leu2-3,112 trpl-1 can 1-100 ura3-1 ade2-1 his3-11,15)] dung de bleu hien protein dung hqp len be met Vector pICAS-1 (PTN.Cong nghe sinh hoc phan dr, Truemg Dai, hoc Khoa hoc tv nhien, DHQGHCM) ducrc thiet ke de lam vector tai to hqp mang gene nisin, co kich tit& 6457bp, mang gene khang ampicilin giup sang 19c d6ng bao vi khan mang vector, gene ma hem protein sinh tong hqp tryptophan giup sang 19c cac &rig nam men mang vector mei trueng khuyet dueng tryptophan, promoter GADPH (glyceraldehyde-3-phosphate dehydrogenease) kiem sat bieu hien beri glucose, trinh tier tiet SSS (secrection signal sequence) giap protein, chuyen vi qua mang, trinh tv gin neo GPI giup On ming to bac), met Oa 3' a-Agglutinin la trinh ter dung h9p yen Nisin Ciu truc vector tai to hqp pICAS-nisin Trinh tv gene nisin duce tei tru hoa cac codon bang phan mem thiet ke gene Snap Gene 2.6.2 (www.snapgene.com) nhArn tucmg thick to bao chit S cerevisiae Gene nisin dtrqc khuech dai bang phin img PCR veri cep mei dee hieu cho gene BglIIN (GGA AGA TCT CCA TGT CTA CTA AAG ATT TTA ATT TGG) va XhoI-N (CCG CTC GAG CGG ATG GTG ATG GTG ATG GTG TTT AGA AAC ATG AAC AGA AC, va duqc cher) vao vector pICAS-1 San pham not duqc hoa bien nap vao to bao E coli DH5a va duce sang 19c tren mei trueng LB agar (trypton 1%, NaCI 1%, cao nam men 0.5%) co khang sinh Ong de 100 µg/ml The bien nap duvc kiem tra bang phan irng PCR khuan lac veri cep mei BglII-N va Plasmid tai to hop duqc kiem tra bing phan img cat veri enzyme Bell va XhoI va kiem tra sv hien dien cua trinh tv gene tren palsmid bang PCR vei cep mei BglII-N va XhoI-N 380 Tao deng bao nAm men S cerevisiae W303 mang vector tai to hqp pICAS-nisin Vector pICAS-nisin duqc hien nap vao to bao nAm men S cerevisiae W303 bang phuang phap lithium acetate (Gietz, Schiestl 2007) San pham bien nap duqc sang 19c tren mei tnreng khuyet clueing tryptophan (SD-: leucin 30mg, Histidin 20mg, Adenin 20mg, Uracin 20mg, Yeast nitrogene base 6.7g, Glucose 20g va ntrac cat du Hit) Cac khuAn lac MQC duqc tren moi tnrerng khuyet duang tien hanh tach chiet geneome (Rose et al., 1990) va PCR kiem tra veri cep mei BglII-N vi Xhol-N dee hieu cho gene nisin De kiem tra sir bleu hien Nisin tren be met to bio nAm men tai to hop, cac chting nAm men tai to hop sau duvc PCR kiem tra tien hanh lai mien djch hujrnh quang(Tang et al., 2008) tren be met to bio veri khang the so cap la khang the don deng khang Histidin tir chuet Anti-His Antibody (GE Healthcare, Thy Si) ti le 1:1000 then gian u gib va khang khang the thin cap la khang the da dor% gin hujmh quang Goat anti-Mouse, Alexa Fluor 488 (Life Technologies, My) ti le 1:3000, thei gian ti gib Cfic bito sau nhuem ducfc quan sat dueri kinh hien vi hujrnh quang btrec song kich thich 488nm KET QUA VA THAO LUAN Toi tru va thiet ke dung hqp gene Mint bieu hi?n Nisin teen be met ban nam men Trong , nghien ciru nay, Nisin duet bieu hien tren be mot to bao nAm men bang dung hop veri met nira cau tnic a —Agglutinin, cAu trite dung hqp gene duce dieu hoa bleu hien bei promoter GADPH (Hinh IA) Theo nghien ciru cua Ueda va Tanaka (2000), a-Agglutinin ding da dirge sir dung film met protein neo de Neu hien protein ngoai lai tren be met to bac) A nam men Nisin la peptide c6 nguen gee tir vi khtffin L lactis, nham tei tru gene ma, hoa cho peptide thong to bio chit nam men, chimg ten sir ding phan mem thiet ke gene Snap Gene 2.6.2 yen be thong so ve codon ma hofi cua nam men S cerevisiae de chuyen el& cac codon kho nhen dien va cac codon khong dich ma tren gene nisin dm L lactis thank cac codon djch ma cho ding amino acid cna nam men Khi so sinh codon ma h6a cho Nisin tir vi khan L lactis vei codon ma hoa cua nAm men S cerevisiae, ket qua cho thay chi co 57% codon Tap chi Cong ngh0 Sinh kw 13(2): 379-385, 2015 ten tong s6 57 codon c6 de ttrcrng thich 100% vai ma di truyen cua nam men S cerevisiae 14% codon c6 de Wang thich tir 50-70% va 28% codon c6 de twang thich nho han 50% (Hinh 1B) Sau tai tru trinh ur gene ma hoa Nisin, ket qua phan tich cho thay 96% codon twang thich 100% voi codon ma hod ctia nam men S cerevisiae, chi c6 4% codon c6 de ttrcmg thich 50-70% (Hinh IC) Trinh tkr neo GPI A Promoter GADPH I SSS Dich ma 411111 W— Neo GPI B Ten gen: Nisin - Lacto Ngu6n g6c: Lactococcus lactis Bang ma so sanh: Saccharomyces cerevisiae Tryc y: m6i tuang quan nh6 han 70% (mau xanh), nh6 han 50% (mau dO) 3333 3 3 mice s s 33g ;33 3333 °S° 333 1 1 1 1 1,1 1 1 1 1 1 118:116FILDLVEVII/LTDSGASTRISISLIPOC VLIIGCALR A CA C4VPIV:.% C Ten gen: Nisin - Yeast Ngu6n g6c: Dirac hen d6i tir Nisin-Lacto Bang ma so sanh: Saccharomyces cerevisiae Tryc y: m6i tuang quan nho han 70% (mau xanh), nh6 han 50% (mau do) :::::::: Ess 33333 333:33333333333333338338333.333333333 too - 11000,111,11101001111 111 1111 so — Hinh Tai vu va thiet ke dung hgp gen nham bieu hien Nisin trOn be mat to bao nem men S cerevisiae (A): Cau trtic gen dung hcrp protein SSS: secrection signal sequence; GADPH: glyceraldehyde-3-phosphate dehydrogenase; GPI: glycosylphosphatidylinositol (B): So sanh bang ma di truyan cue trinh ti,r gen nisin ma hda tit, L lactis \Idyl nam men S cerevisiae (C): So earth bang ma di truyan sau tai trinh tio, gen nisin vai nam men S cerevisiae 381 Nguyen Hieu Nghia et al Cau frac vector tai to hop pICAS-nisin Gene nisin duqc thu nhan bang phan Cmg PCR, sau dtrqc cat bang hai enzyme cat giai hen BgiII va •XhoI Ket qui dien di tren gel agarose 1%, san phi= PCR cho mOt vach kich thu6c khoang 200bp nhu da thiet ke (Hinh 2A, gieng 3) Song theo do, vector pICAS-1 cling duqc cat ma wing bang hai enzyme BglII va Xhol cho mOt vach c6 kich thu6c khoing 6500bp tren ban dien di (Hinh 2B, gieng 4) San phim gene va plasmid sau cat ducc not lei va Wen nap vao to bao E colt DH5a Cac d6ng bao E colt DH5a tai to hop duqc sang loc tren moi tnrang LB agar ampicilin va kiem tra bang PCR khuan lac vai cap mai Bg1117N va Xhol-N dac hieu cho gene nisin Ket qua kiem tra cho they mOt so clang to bao E coil cho san pham A PCR mOt vach twang ducmg chimg ducmg (Hinh 2B, gieng 2, giong 3, gieng 5-7), dial d6 chimg to cac thing to bac) mang gene mac heti BEn canh do, cac plasmid tai to hqp tir cac dong tai to hqp duqc PCR vai cap moi Bg111-N/XhoI-N cling cho san pham PCR twang to chimg duang (Hinh 2C, gieng 2-4) Cu6i ding, cac plasmid tai to hqp duqc cat bang hai enzyme Bg111 va Xhol va giai trinh hr d8 khing dinh trinh ti,r cling nhu si,r khung dich ma Ket qua dien di san pham cat cho they plasmid tai to hop sau cat bang hai enzyme da cho hai vach, mOt vach co kich thu6c khoing 6500bp tucmg ducrng kich plasmid pICAS-1 duqc cat va mOt vach co kich thu6c khoang 200bp ttrang, dung kich thuac gene nisin (Hinh 2D, gieng 2, gieng 3) Nhu vay, plasmid tai to hcp pICAS- nisin de duqc too ding by 6000 — 3000 — 1000 — 250 — C by 6000 — 3000 — 1000 — 250 — D by 6000 — 3000 — 1000 — 250 — Hinh Chen gen nisin vao vector pICAS1 va kiem tra vector tai t6 hap pICAS-nisin (A): Thu nhan gen nisin va plasmid pICAS-1 Thang DNA kbp; Chung am phan Ong PCR; San pham PCR gen sau cat; Plasmid sau cat; Plasmid Utah bang phvang phap SDS kiem (B): PCR khan lac E coil DH5a/pICAS-nisin Thang DNA kbp; 2-7 San pham PCR khan IOC E coil DH5a khac nhau; ChOng am phan Ong PCR; Chirng &rang phan Ong PCR (C): PCR plasmid tai td hap E coil DH5a/pICAS-nisin Thang DNA kbp; 2-4 San pham PCR plasmid khac nhau; Chirng dtrang phan Ong PCR; Chirng am phan Ong PCR (13): Cat plasmid tai t6 hop pICAS-nisin bang BgllI va Xhol Thang DNA kbp; 2-4 San pham cat plasmid khac nhau; Plasmid pICAS-1 382 Tap chi Cong nghe Sinh hac 13(2): 379-385, 2015 B A Scarevistao W303 SD- (Trp-) Tryptophan SD- (Trp-) C S.C.enovislas W303/ pICAS-nisin D nisin 6xHistidine Promote GPI Xhol-N us , Wit nil a-agglutinin IPCR Pha tai Pha sing S.Convisloo W303 600030001000- 250— Pha sang Pha tit s.centvislas W3031pICAS-nisin Hlnh Sang loc clang to bao nem men mang vector tai t6 hqp pICAS-nisin (A): Nguyen tic sang lqc tren mei trtreng khuyet clueing (B): Ket qua Wen nap vector pICAS-nisin vao to bao nem men S cerevisiae W303 (C): Kiem tra skr hien dien die dung hop gen to bao nem men Thang DNA 100bp; Kik qua phan irng PCR v6i khulen la DNA be gen cua nem men; Dung hqp gen; 4-9 Keit qua phan itng PCR v&i khu6n la DNA be gen tit cac khuan lac nem men tai 16 hqp sang loc dttqc tren mei trtteng (D): Kiem tra bleu hien gen nisin tren be met te bao bang nhuem mien djch huynh quang yeti khang the khang Histidin Pha sang le anh sang trAng va pha t6i anh sang huynh quang bulfrc song 488 nm Tao dung t4 bite nem men S cerevisiae W303 thi nghie m chuyen dung hqp gene nisin - a - mang vector di to hqp pICAS-nisin ye bieu hien Nisin tren be mat to bao agglutinin va bie.0 hien Nisin ten be mat to bao la to bao nem men S cerevisiae W303 mang d6t bien re bao cha nem men S cerevisiae ductc sir dung khuyet &rang tryptophan, khang phat trien dtrqc tren mai trtremg SD- (yeast minimal media minus 383 Nguyen Hieu Nghia et al tryptophan) 'Chi bio dim men W303 tiep nh#n vector tai to hqp pICAS-nisin, trinh to gene ma hem protein sinh tong hqp tryptophan tren vector duqc sat nh#p vio bil) gene ciut nam men, v#y d6ng NW co nang sinh twang tren moi tru6ng SD" (Hinh 3A) Cic ket qui time nghiem cia chum; t8i cho thiy, sau bien nap vector pICAS-nisin van bao nam men S cerevisiae W303, chin toi di thu duqc mgt so khuAn lac nam men phit trien tren m8itru6ng SD" (Hinh 3B) Cic deng bac) nam men tai to hqp tiep tVc duqc kiem tra su tai to hop cis gene nisin vac) b0 gene nam men Ket qua cho thAy 5/30 deng nam men phit trien duqc tren m6i truing SD", da duqc chen gene nisin vio b0 gene the hien qua lc& qui PCR duang tinh sir dting DNA b0 gene cis cfic deong nam men lim khu8n mau phan 1mg PCR veri cep mOi d#c hieu cho gene nisin (Hinh 3C, gieng 4-7) Nhu trinh bay tren hinh 3C, gene nisin duqc ket hqp them codon ma hoi cho cic amino acid histidine tram duqc dung hqp veri mCit nira cau trite a-Agglutinin Viec dung hqp veri the histidine nham mtic dich nh#n dien chat peptide Nisin tren be m#t to bao dim men thong qua phuang thirc nhuOrn mien dich huynh quang veri khing the khing histidine icet qua thuc nghiem cho they dual inh sing huynh quang, &mg to bao S cerevisiae W303/pICAS-nisin cho tin hien huynh quang teen be met bio Tin hieu kh8ng quan sit ductc tren deng to bao S cerevisiae W303 (Hinh 3D) Nisin vi vi sinh sinh tong hqp Nisin da va clan§ duqc sir ding rOng di b nhieu clang Ichic nhau: Che phi= probiotic hay clang peptide khing khuin (Deegan et al., 2006) Nhieu nghien cim truck day di cling ve ve cic ket qua bieu hien Nisin tai to hqp tren cic he thOng bao chu L lactis (Mierau, Kleerebezem, 2005) Tuy nhien, Nisin tai hqp thutmg duvc tong hop va tiet ben ngoii mei truemg Vai ham luqng bleu hien kh8ng cao, viec thu nhan nisin tir m8i truemg nuoi ciy slip khong it kh6 khan va la nguyen nihan lam giim hieu qua cia quit trinh sin xuat nisin tai to hq? Nghien clru cis chang toi la nghien ciru ditu tien bieu hien nisin tai tto hop hang thii dinh tren be met to bio nAm men Ket qua thuc nghiem khing djnh chimg Vol di cling viec bieu hien Nisin be met to bio nam men S cerevisiae 384 KET LUAN Trong nghien ciru nay, chimg toi da cAu true cong vector tai to hop pICAS-nisin nham bieu hien Nisin dung hqp 3'a-Agglutinin tren be met to bao nAm men S cerevisiae Dong theri biz& du sang lqc duqc deng bao am men S cerevisiae W303 mang vector pICAS-nisin Dong dim men tai to hqp li nguyen lieu ca bin cho thir nghiem hoot tinh Nisin duqc dung hqp vi c0 djnh tren ming to bao, cAng nhu nghien cuu (mg ding chung bio quirt thuc phew vi probiotic TAI LI$U THAN' KHAO Basanta A, Herren C, Gutierrez J, Criado R, Herandez PE, Cintas LM (2009) Development of bacteriocinogeneic strains of Saccharomyces cerevisiae heterologuously expressing and secret the leaderless EnterocinL50A and L5OB from Enterococcus faecium L50 App! 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Microbiol Biotechnol 94: 1061 - 1068 Deegan LH, Cotter PD, Hill C, Ross P (2006) Bacteriocins: biological tools for bio-preservation and shelf-life extension Int Dairy J 16: 1058-1071 Fukao M, Obits T, Yoneyama F, Kohda D, Zendo T, Nakayama J, Sonomoto K (2008) Complete Covalent Structure of Nisin Q, New Natural Nisin Variant, Containing Post-Translationally Modified Amino Acids Biosci Biotechnol Biochem 72: 1750- 1755 Gietz RD, Schiestl RH (2007) High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method Nat Protoc 2: 31-34 Klaenhammer TR (1993) Genetics of bacteriocins produced by lactic acid bacteria FEMS Microbiol Rev 12: 39-85 Mierau 1, Kleerebezem M (2005) 10 year of nisincontrolled gene expression system (NICE) Appl Microbiol Biotechnol 68:705-717 Piper C, Draper LA, Cotter PD, Ross RP, Hill C (2009) A comparison of the activities of lactacin 3147 and nisin against drug-resistant Staphylococcus aureus and Tap chi Cong nghe Sinh hoc 13(2): 379-385, 2015 Enterococcus species J Antimicrob Chemother 64: 546-551 Reenen CAV, Chikindas ML, Zyl WHV, Dicks LMT (2003) Characterization and heterologous expression of a class Ifa bacteriocin, Plantaricin 423 from Lactobacillus plantarum 423 in Saccharomyces cerevisiae Int J Food Microbiol 81: 29-40 Rose MD, Winston F, Hieter P (1990) Methods in Yeast Genetics A Laboratory Course Manual Cold Spring Harbor, NY Schoeman H, Vivier MA, Toit MD, Dicks LM, Pretorius IS (1999) The development of bactericidal yeast strains by expressing the Pediococcus acidilactici Pediocin Gene (pedA) in Saccharomyces cerevisiae Yeast 15: 647-656 Tang YQ, Han SY, Hong ZH, Wu L, Ueda M, Wang XN, Lin Y (2008) Construction of cell surface-engineered yeasts displaying antigene to detect antibodies by immunofluorescence and yeast-ELISA Appl Microbiol Biotechnol 79: 1019-1026 Ueda M, Tanaka A (2000) Genetic immobilization of proteins on the yeast cell surface Biotechnol Adv 18: 121-140 Yang SC, Lin CH, Sung CT, Fang JY (2014) Antibacterial Activities of Bacteriocin Application in Foods and Pharmaceuticals Front Microbiol 5: 1-10 Zendo T, Fukao M, Ueda K, Higuchi T, Nakayama J, Sonomoto K (2003) Identification of the Lantibiotic Nisin Q, a New Natural Nisin Variant Produced by Lactococcus lactis 61 - 14 Isolated from a River in Japan Biosci Biotechnol Biochem 67: 1616-1619 CLONING AND EXPRESSION NISIN ON SACCAROMYCES CEREVISIAE CELL SURFACE BY FUSION WITH a-AGGLUTININ GENE Nguyen Hien Nghia, Thai Ngoc Anh, Nguyen Quynh Huong, Ngo Thi Cam Nhung, Nguyen Tri Nhan, Tran Linh Thuoc, Dang TM Phuong Thao23 University of Science, Vietnam National University in Ho Chi Minh SUMMARY Nisin is a bacteriocin which has a board antibacteria spectrum and inhibiting pathogeneic bacteria in humans and animals, such as Staphylococcus aureus, Listeria sp In addition, althought Nisin was obtained from fermenttion broth of Lactococcus lactis that have been widely applied in food preservation industry, but native strains secreted naturally Nisin with low levels Hence, the domestic and foreign researches currently perform to increase Nisin expression level by recombinant techniques in many host cells as Escherichia coli or Saccharomyces cerevisiae (S cerevisiae) In order to find a new approach to applications in food preservation as well as collect new strains inhibiting pathogeneic bacteria in cattle and aquaculture We will show strategy in which we aim to express Nisin on S cerevisiae cell - surface by fusion nisin gene with a-agllutinin gene The yeast cell lines are collected as the raw material for applied research in preservation food as well as in the preparations inhibiting pathogeneic microorganisms Keywords: Agglutinin, bacteriocin, fluoresent, nisin, pICAS-1, Saccharomyces cerevisiae, yeast surface 23 Author of correspondence: E-mail: thaodp@hcmus.edu 385 ... nm Tao dung t4 bite nem men S cerevisiae W303 thi nghie m chuyen dung hqp gene nisin - a - mang vector di to hqp pICAS -nisin ye bieu hien Nisin tren be mat to bao agglutinin va bie.0 hien Nisin. .. hop pICAS -nisin nham bieu hien Nisin dung hqp 3'a -Agglutinin tren be met to bao nAm men S cerevisiae Dong theri biz& du sang lqc duqc deng bao am men S cerevisiae W303 mang vector pICAS -nisin Dong... ke dung hgp gen nham bieu hien Nisin trOn be mat to bao nem men S cerevisiae (A): Cau trtic gen dung hcrp protein SSS: secrection signal sequence; GADPH: glyceraldehyde-3-phosphate dehydrogenase;