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NGHIN CU CU TRC V SNG LC DNG NM MEN Pichia pastoris

TI T HP A BN SAO BIU HIN NHN T TNG TRNG T TIU CU (BB-PDGF-BB) MC CAO

Vng Ct Khnh, Ng Th Huyn Trang, Nguyn Phm Phng Thanh,

ng Th Phng Tho*, Trn Linh Thc

Trng i hc Khoa hc t nhin tp H Ch Minh, *thaodp@hcmus.edu.vn

TM TT: Nhn t tng trng t tiu cu PDGF-BB (Platelet derived growth factor) c t chc FDA-Hoa K (Food and Drug Administration) ph chun l sn phm s dng trong iu tr chng lot chn do i tho ng. Trong nghin cu ny, chng ti to dng thnh cng cc chng nm men ti t hp mang gen pdgf v sng lc c 2 dng Pichia pastoris ti t hp mang nhiu bn sao gen pdgf. Cc dng ti t hp a bn sao gen pdgf sau khi sng lc c kim tra v so snh kh nng biu hin PDGF vi dng n bn sao. Kt qu cho thy kh nng biu hin PDGF tng theo s lng bn sao gen mc tiu st nhp vo trong b gen. Chng a bn sao thu nhn c c kh nng sn xut lng PDGF chim trn 50% tng lng protein tit ra mi trng. T kha: Pichia pastoris, dng a bn sao, i tho ng, PDGF-BB ti t hp.

M U

PDGF (Platelet derived growth factor) l mt yu t phn bo ch yu ca nguyn bo si, t bo c trn v nhiu t bo khc, c sn xut t tiu cu. PDGF gi nhiu chc nng trong c th, c bit l chc nng tham gia iu ha v thc y qu trnh lm lnh vt thng [6]. Protein ny tc ng n nhiu t bo lin quan n qu trnh hn gn vt thng nh: kch thch s phn bo v hng ha ca nguyn bo si v t bo c trn, kch thch i thc bo sn xut v tit nhng nhn t tng trng quan trng, kch thch s sn sinh nhng phn t fibronectin, collagen, proteoglycan v hyaluronic acid. Trong giai on ti to, PDGF c vai tr kch thch sn xut v tit collagenase bi nguyn bo si cng nh s co cc si collagen [1]. Vi s gia tng bnh i tho ng nh hin nay, t l bnh nhn mc chng lot chn ngy cng tng. Tuy nhin, ti Vit Nam, ngun cung cp PDGF ch yu l t nc ngoi vi gi thnh cao. iu ny t ra yu cu cp thit trong vic nghin cu cng ngh sn xut PDGF nhm pht trin sn phm thuc iu tr c hiu qu vi gi thnh hp l.

Trong s cc h thng biu hin protein ngi ti t hp, Pichia pastoris c xem nh mt h thng ni bt vi nhiu u im nh d nui cy, tng trng nhanh, protein c bin

i sau dch m. Bn cnh cc u im trn, h thng ny c tnh bn vng di truyn qua nhiu th h t bo do vector ti t hp mang gen mc tiu c th c st nhp vo b gen nm men theo c ch ti t hp tng ng gia DNA plasmid v vng trnh t tng ng trn b gen theo 2 cch: (1) gn chn vector vo b gen nm men hoc (2) thay th gen, loi b hon ton gen AOX1 trong b gen nm men. S lng bn sao ca gen mc tiu st nhp vo b gen chng ch nm men c th l mt trong cc yu t gip gia tng mc biu hin protein mc tiu. Hn na, protein ngoi lai c tit ra ngoi mi trng dng c hot tnh, v th, qu trnh thu nhn, tinh ch protein d dng hn so vi protein c biu hin dng ni bo. Ngoi ra so vi Saccharomyces cerevisiae protein c biu hin Pichia pastoris khng b ng ha qu mc v khng hnh thnh lin kt -1,3 glycan, lin kt ny c cho l gy ra p ng min dch khi dng trong tr liu [2, 4, 5]. VT LIU V PHNG PHP NGHIN CU

Vt liu plasmid pPICZA (V195-20, Invitrogen); chng E. coli DH5 [F-, 80lacZM15, recA1, endA1, hsdR17(rk-, mk+), phoA, supE44, -, thi-1, gyrA96, relA1] (Invitrogen) c cung cp bi B mn Cng ngh sinh hc phn t v Mi trng, Trng

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i hc Khoa hc t nhin, i hc Quc gia thnh ph H Ch Minh; chng Pichia pastoris X33 (C180-00, Invitrogen). To dng t bo E. coli DH5 mang plasmid biu hin PDGF-BB (PDGF)

Gen pdgf c thu nhn bng phng php PCR s dng cp mi c hiu FXhoI v RnotI. Sn phm PCR (357 bp, m ha cho chui polypeptide di 119 acid amin vi khi lng phn t l 17 KDa) sau khi tinh sch c x l vi enzyme XhoI v NotI, sau ni vo vector pPICZA cng c ct m vng bng 2 enzyme trn. Plasmid ti t hp pPICZA/pdgf c bin np vo t bo E. coli DH5 bng phng php ha bin np, s dng yu t chn lc l kh nng khng khng sinh zeocine.

Plasmid ti t hp pPICZA/pdgf c kim tra s chn gen pdgf bng k thut PCR vi cp mi FXhoI v 3AOX, enzyme ct gii hn XhoI v NotI, gii trnh t. To dng t bo Pichia pastoris X33 mang gen m ha PDGF

Plasmid ti t hp pPICZA/pdgf c thu nhn t chng E. coli DH5 bng phng php SDS kim, sau x l bng enzyme SacI ct m vng, in bin np vo t bo Pichia pastoris (P. pastoris) X33 v c nui cy trn mi trng YPD (c thnh phn gm cao nm men 1%, peptone 2%, dextrose 2%, agar 2% [7]) c nhn t chn lc l zeocine 100 g/ml, 25oC, 3-5 ngy. Kim tra kiu hnh, kiu gen ca cc th bin np

Kim tra kiu hnh: cc th bin np c cy ng thi ln hai mi trng MM (mi trng ti thiu cha methanol c thnh phn YNB 1,34%, biotin 4.10-5%, methanol 0,5%) v MD (mi trng ti thiu c cha dextrose c thnh phn YNB 1,34%, biotin 410-5%, glucose 2%), sau em 25oC trong 3-4 ngy v quan st.

Kim tra kiu gen: b gen ca cc th bin np c tch chit v kim tra kiu gen bng phn ng PCR vi cp mi 5AOX1 v 3AOX1. Xc nh s bn sao ca vector mang gen trong b gen ca P. pastoris bng phng php PCR bn nh lng

B gen ca cc th bin np sau khi pha long trong khong 6.255 ng - 800 ng c dng lm khun cho phn ng PCR vi cp mi 5AOX1 v 3AOX1 trong 20 chu k. Kt qu khuch i c in di trn gel agarose 1% v phn tch bng phn mm ImageJ nhm xc nh s bn sao gen pdgf trong b gen nm men. S bn sao gen pdgf trong b gen nm men c tnh thng qua t l sn phm khuch i cha gen pdgf so vi sn phm khuch i gen AOX1, mt gen ni sinh ca nm men. Cc kt qu phn tch c x l bng phm mm ImageJ (http://imagej.nih.gov/ij/download.html) nh gi kh nng tng trng v biu hin ca cc th bin np cha nhiu bn sao

Mt khun lc n X33, X33 cha pPICZA v cc th bin np mang gen pdgf c hot ha trong mi trng BMGY lng (c thnh phn gm cao nm men 1%, peptone 2%, YNB 1,34%, 10% citrate buffer 1 M pH 4,0; biotin 410-5%, glycerol 1% [7]), nui cy lc qua m 250 vng/pht, 300C n khi OD600 ca dch nui cy t 2-6. Sau sinh khi c chuyn vo mi trng BMMY (c thnh phn nh mi trng BMGY nhng glycerol c thay th bng methanol c nng cui l 0,5% (v/v)) v tip tc nui cy lc. Sau mi 24 gi, b sung methanol t nng cui 0,5%, ng thi dch nui cy c thu nhn, o OD600 nh gi kh nng tng trng. Qu trnh cm ng biu hin cc th bin np trn mi trng BMMY pH 4,0 c thc hin tng t nh trn, 72 gi sau cm ng, dch nui cy c ly tm 13000 vng/pht, trong 5 pht, sau loi sinh khi, thu nhn dch mi trng. S biu hin protein PDGF c kim tra bng phng php in di trn gel SDS-PAGE, phn tch bng phn mm Quantity One ((Biorad, http://www.bio-rad.com/en-us/product/quantity-one-1-d-analysis-software) v lai Western

KT QU V THO LUN

To dng t bo E. coli DH5 mang plasmid biu hin PDGF

Plasmid ti t hp pPICZA/pdgf sau khi to thnh c bin np vo t bo E. coli DH5. Cc th bin np sau khi sng lc trn mi

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trng khng sinh zeocine c tip tc kim tra bng phng php PCR khun lc s dng cp mi 5AOX1 v 3AOX1. Cc th bin np cho kt qu PCR khun lc dng tnh (xut hin 1

vch c kch thc vo khong 870 bp trn bn in di) c tip tc tch chit thu nhn plasmid ti t hp bng phng php SDS-kim (kt qu khng c trnh by y).

Hnh 1. Sng lc vector ti t hp mang gen pdgf

a. Sng lc vector ti t hp bng phn ng PCR; a1. V tr mi AOX1 trn b gen nm men ti t hp; a2. Kim tra vector ti t hp bng PCR; b. Kim tra vector ti t hp mang gen pdgf bng phng php ct gii hn; b1. V tr enzyme ct gii hn trong vector; b2. X l vector ti t hp bng enzyme ct gii hn.

Nhm pht hin gen pdgf trn plasmid

pPICZA/pdgf, plasmid ti t hp c phn tch bng phng php s dng cp mi c hiu FXhoI (mi xui) trn gen v 3AOX1 (mi ngc) trn plasmid. Nhng plasmid pPICZA c chn gen pdgf s cho vch DNA vi kch thc khong 540 bp, tng ng vi vi kch thc vch gen pdgf cng vi mt on DNA trn plasmid pPICZA. Trong khi , vi khun l plasmid pPICZA khng mang gen pdgf nn khng c sn phm PCR (hnh 1a).

Kt qu ct kim tra plasmid ti t hp pPICZA/pdgf bng enzyme XhoI v NotI cho 2 vch ng nh d kin: mt vch c kch thc tng ng vi kch thc ca plasmid pPICZA, v mt vch c kch thc tng ng vi gen pdgf (hnh 1b).

ng thi, kt qu gii trnh t gen pdgf trn plasmid pPICZA/pdgf bng cp mi 5AOX1 v 3AOX1 sau khi so snh vi trnh t l thuyt do chng ti thit k v ci bin cho gen pdgf biu hin trong nm men bng phn mm Jellyfish (http://www.cbcb.umd.edu/ software/jellyfish/) cho tng ng 100%.

iu ny cho php kt lun chng ti to dng ha thnh cng gen pdgf vo vector biu hin pPICZA trong t bo E. coli DH5. To dng t bo P. pastoris X33 mang gen m ha PDGF

Plasmid pPICZ/pdgf c a v dng thng bng cch x l vi enzyme SacI v in bin np vo t bo ch P. pastoris X33. Cc th bin np sau khi sng lc da trn kh nng khng khng sinh zeocine c kim tra kiu hnh v kiu gen.

Hnh 2. Kt qu kim tra kiu hnh nm men

mang gen pdgf

a b

MM MD

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Khi DNA plasmid c st nhp vo b gen ca t bo ch P. pastoris trong qu trnh ti t hp tng ng, gen AOX1 (m ha cho enzyme alcohol oxidase, enzyme chnh quy nh kh nng s dng methanol ca P. pastoris) c th gi li hoc b loi b, v vy kiu hnh ca th bin np s thay i. Nu gen AOX1 khng b mt i, kiu hnh cc th bin np thu c l Mut+, c kh nng tng trng tt trn mi trng methanol. Nu gen AOX1 b loi b, th bin np s c kiu hnh MutS sinh trng km trn mi trng c methanol.

Kt qu so snh s pht trin ca th bin np trn hai mi trng MD v MM, cho thy tt c cc th bin np u tng trng tt trn c hai mi trng (hnh 2). Do , tt c cc th bin np u c kiu hnh Mut+ (cn gi li gen AOX1 trong b gen).

xc nhn kt qu kim tra kiu hnh (hnh 2), DNA b gen ca cc th bin np

c tch chit v thc hin phn ng PCR kim tra kiu gen bng cp mi 5AOX1 v 3AOX1. Sn phm PCR t cc chng Mut+ khi in di s thu c 2 vch. Mt vch c kch thc 875 bp tng ng kch thc tng ca gen pdgf v vng gen AOX1 nm trn plasmid. Vch cn li c kch thc khong 2,2 kb, tng ng vi gen AOX1 c sn trong b gen ca X33. cc chng MutS th khng c vch 2,2 kb ny.

Kt qu PCR cho thy tt cc cc dng u xut hin 2 vch trn bn in di (hnh 3), iu ny chng t tt c cc th bin np u c kiu hnh Mut+, ph hp vi kt qu kim tra kiu hnh. Cc dng ny tip tc c phn tch nhm xc nh s lng pPICZ/pdgf c st nhp vo b gen bng phng php PCR bn nh lng. Xc nh s lng bn sao ca gen pdgf trong b gen cc th bin np

Hnh 3. PCR kim tra kiu gen cc th bin np. (a) V tr bm ca mi AOX1

v cc kt qu PCR d kin tng ng vi kiu hnh Mut+ v Muts; (b) Kt qu PCR kim tra kiu gen cc th nm men mang gen pdgf

i vi cc th bin np nm men kiu hnh

Mut+, khi thc hin phn ng vi mi AOX1 (hnh 3) bn cnh sn phm PCR l gen AOX1 c kch thc 2,2 kb, phn ng cn cho sn phm DNA dung hp cha gen mc tiu pdgf di khong 875 bp c ngun gc t plasmid ti t hp v c st nhp vo b gen ca t bo nm men trong qu trnh bin np. S lng cc bn sao DNA chn vo b gen l hon ton ngu nhin. Nu b gen th nm men bin np cha 1 bn sao DNA dung hp, lng sn phm DNA 875 bp cha gen pdgf s tng tng vi lng sn phm khuch i t gen AOX1 ni sinh (2,2 kb). B gen th bin np nm men cha cng

nhiu bn sao ca gen pdgf th t l sn phm khuch i cng tng so vi gen AOX1 ni sinh. Bng cch tnh t l sn phm khuch i, chng ti xc nh tng i s lng bn sao pdgf trong th nm men bin np (hnh 4). Kt qu cho thy, trong 20 th nm men mang gen pdgf thu c, c 2 chng mang nhiu bn sao gen pdgf hn so vi cc chng cn li (1 chng mang 16 bn sao v 1 chng mang 4 bn sao gen pdgf so vi cc chng cn li ch mang 1 bn sao ca gen pdgf). Chng ti tin hnh ghi nhn kt qu v kho st kh nng tng trng cng nh kh nng sinh tng hp PDGF ca cc chng nm men ny.

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Hnh 4. PCR bn nh lng xc nh s lng bn sao ca cc th bin np

a. V tr mi AOX1 trn b gen Mut+; b. Kt qu in di sn phm PCR bn nh lng; c. S lng bn sao tng i ca cc th bin np

Hnh 5. ng cong tng trng ca chng

X33 hoang di v cc th bin np nh gi kh nng tng trng ca cc th bin np nm men a bn sao pdgf

Vic cha nhiu bn sao ca vector trong b gen c th s nh hng ti sc sng ca cc

th bin np. Kt qu kho st tng trng cho thy kh nng tng trng ca cc th bin np khng khc bit nhiu so vi chng X33 hoang di, gi tr OD600 cao nht u t khong 10-15 sau 48 gi cm ng (hnh 5). iu ny chng t cc th bin np a bn sao thu nhn c khng b gim sc sng do hin tng st nhp gen gy ra. Kim tra kh nng biu hin PDGF ca cc th bin np nh gi s nh hng ca s lng bn sao

gen st nhp n kh nng biu hin protein mc tiu, chng ti tin hnh cm ng biu hin

PDGF t cc th bin np thu nhn c.

Hnh 6. Kh nng biu hin PDGF ca cc th bin np

a. SDS-PAGE iu kin bin tnh; b. PAGEiu kin khng bin tnh; c. %PDGF/tng protein tit.

Kt qu kim tra protein bng SDS-PAGE cho thy, dch nui cy ca cc th bin np (hnh 6) xut hin 1 vch protein c kch thc trong khong 17 kDa trong iu kin bin tnh tng ng vi kch thc PDGF monomer v 31 kDa trong iu kin khng bin tnh tng ng vi PDGF cu hnh dimer, trong khi nm

men P. pastoris khng chn gen pdgf khng sn xut c protein ny. Phn tch bng phn mm Quantity One chng ti nhn thy vch protein ny m v nhiu hn hn th bin np a bn sao (th 1 v 4) v nhiu nht th bin np 1, chim 44,5% so vi tng lng protein tit ca t bo (hnh 6c). Kt qu ny

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cng ph hp vi d on t kt qu kim tra s lng bn sao pPICZA/pdgf trc . Dng ti t hp 1 vi s lng bn sao pdgf st nhp nhiu nht (16 bn sao) c kh nng biu hin protein mc tiu mc cao nht. Th v hn na, khi chng ti tin hnh in di protein thu c t nm men trn gel polyacrylamide khng b sung tc nhn bin tnh (hnh 6b), chng ti nhn thy protein tit thu c cc chng nm men c kch thc khong 31 kDa. Theo cc nghin cu ca Raines (1982) [8] v

Wang (2008) [9] y c th l trng thi dimer ca protein PDGF. iu ny cho thy kh nng thu nhn protein PDGF trng thi dimer (l trng thi c hot tnh sinh hc) t cc chng nm men ti t hp. Dng ti t hp 1 c tip tc s dng phn tch kh nng biu hin protein PDGF theo thi gian. Dch cm ng c thu nhn sau 24, 48 v 72 gi, sau in di SDS-PAGE v lai western vi khng th c hiu khng PDGF (AB20NA, R&D) (hnh 7).

Hnh 7. Kh nng biu hin PDGF theo thi gian ca th bin np 1

a. SDS-PAGE; b. lai Western vi khng th khng PDGF; c. % PDGF trn tng protein tit.

Kt qu trn bn phim cho thy, dch nui cy ca cc th bin np 1 sau 24 gi, 48 gi v 72 gi cm ng u xut hin mt bng sng duy nht tng ng vi v tr ca bng protein kch thc khong 31 kDa trn hnh PAGE. iu ny gip khng nh vch 31 kDa quan st trn bn in di chnh l protein PDGF dng dimer.

Phn tch cc vch protein mc tiu bng phn mm Quantity One, chng ti nhn thy trong 72 gi nui cy, hiu qu biu hin v tit PDGF ca th bin np 1 ngy cng tng. Ti thi im 72 gi, protein PDGF c tit ra chim ti 55,6% so vi tng protein tit ca t bo.

KT LUN

Vi cc kt qu thu c, chng ti kt lun thu nhn c 2 dng nm men ti t hp a bn sao gen pdgf. Cc dng nm men mang nhiu gen chn ny c kh nng sinh trng bnh thng nh chng ch nm men X33 ban u v c kh nng biu hin protein PDGF. Trong , dng Pichia pastoris X33: pdgf mang 16 bn sao gen pdgf c kh nng biu hin v

tit PDGF cao, chim 55,6% tng lng protein tit. Cc kt qu ny l tin cho cc nghin cu pht trin sn phm protein PDGF ti t hp tip theo.

TI LIU THAM KHO

1. Berlanga-Acosta J., Gavilondo-Cowley J., Barco-Herrera D. G., Martn Machado J., Guillen-Nieto G., 2011. Epidermal growth factor (EGF) and platelet-derived growth factor (PDGF) as tissue healing agents: clarifying concerns about their possible role in malignant transformation and tumor progression. Journal of Carcinogenesis & Mutagenesis, 1:115. doi:10.4172/2157-2518.1000115

2. Darby R. A., Cartwright S. P., Dilworth M. V., Bill R. M., 2012. Which yeast species shall I choose? Saccharomyces cerevisiae versus Pichia pastoris (review). Springer, 866: 11-23.

3. Embil J. M., Papp K., Sibbald G., Tousignant J., Smiell J. M., Wong B., Lau C. Y., 2000. Recombinant human platelet-

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derived growth factor-BB (becaplermin) for healing chronic lower extremity diabetic ulcers: an open-label clinical evaluation of efficacy. Wound Repair and Regeneration: Official Publication of the Wound Healing Society [and] the European Tissue Repair Society, 8(3): 162-8

4. Grinna L. S., Tschopp J. F., 1989. Size distribution and general structural features of N-linked oligosaccharides from the methylotrophic yeast, Pichia pastoris. Yeast, 5: 107-115.

5. Hamilton S. R., Davidson R. C., Sethuraman N., Nett J. H., Jiang Y., Rios S., Bobrowicz P., Stadheim T. A., Li H., Choi B-K., Hopkins D., Wischnewski H., Roser J., Mitchel T., Strawbridge R. R., Hoopes J., Wildt S., Gerngross T. U., 2006. Humanization of yeast to produce complex

terminally sialylated glycoproteins. Science, 313: 1441-1443.

6. Heldin C. H., Westermark B., 1999. Mechanism of action and in vivo role of platelet-derived growth factor. Physiol. Rev., 79(4): 1283-316.

7. Pichia expression kit, Invitrogen, 2010, Catalog no. K1710-01.

8. Raines E. W., Ross R., 1982. Platelet-derived growth factor. J. Bio. Chem., 257(9): 5154-5160.

9. Wang Y., Xue, L., Li Y., Zhu Y., Yang B., Wang X., 2009. High-level secretory production of recombinant human platelet-derived growth factor-BB by Saccharomyces cerevisiae under the non-selective conditions. Appl. Biochem. Microbiol., 45(2): 154-161.

A STUDY ON CONSTRUCTING AND SCREENING OF MULTI-COPY

pdgf RECOMBINANT Pichia pastoris CLONES PRODUCING HIGH YIELD OF PDGF-BB (Platelet derived growth factor BB)

Vuong Cat Khanh, Ngo Thi Huyen Trang, Nguyen Pham Phuong Thanh,

Dang Thi Phuong Thao, Tran Linh Thuoc Vietnam National University, Ho Chi Minh city, University of Science

SUMMARY

Platelet-derived growth factor-BB has been approved by US Food and Drug Administration-FDA for the treatment of neuropathic ulcers. Since the number of diabetic patients has increased rapidly, it is necessary to study the production of PDGF-BB with low cost for treatment. Among the expression systems used for human recombinant protein expression, Pichia pastoris is the remarkable one with many advantages. Multiple integrated copies of target gene may give advance in driving up expression level of target protein. In this study, we succeeded in constructing and screening two yeast clones, which carry multi-copy of pdgf target gene. The two multi-copy yeast clones were subjected indetermination of PDGF expression level and compared to single-copy yeast clones. Our results showed that multi-copy pdgf yeast clones have higher level expression of target protein. PDGF produced by the multi-copy pdgf yeast clones accounted for over 50% of total secretory protein, and was folded in dimer form, which is necessary for performing biofunction in wound healing. Keywords: Pichia pastoris, multi-copy clone, platelet-derived growth factor BB, yeast. Ngy nhn bi: 15-7-2013