HOI NGH! KHOA HOC CONG NGHE SINH HOC TO^ ••-' -•
TAO DONG VA Bieu HIEN GENE MA H 6 A LUMBROKINASE d PICHIA PASTORIS
Vu Thj Bich Ngpc, Ly Thi Bich Thuy, Quyen Dinh Thi
Vi^n Cdng nghd sinh hgc, Vi^n Han Idm Khoa hgc vd Cdng ngh$ Vi#f Nam T 6 M TAT
Tic nghSn m^ch U tinh trang u dong m^ch miu vdi cyc m4u dfing, co thi dfin tdi nhoi mau co tim va dgt quy do thieu mau cgc bg, ci hai d k c6 the din tdi tir vong. E)6i voi die trudng bgp cjp tinh, phuong phip di6u tri cd thS la phau thuat ISy cac cyc mau dong hofc thong tic mach miu. Tuy nhiSn, nhung truong hpp man tinh hoac c6 nguy co thi cin phai dilu tn lau dai bin^ thulc ph6ng chong tic nghen m^ch. Lumbrokinase la mflt nhom cic enzyme c6 hoat tinh thiiy phan fibnn cao tit giun dat, c6 khoi lugng phan oi -25-40 L-n.. /-*£.. »..*...»A ..!:.. .43 A.tn^ /.\.ima minh vTrg i^n ttia ngna ttii'iv nlinn frin^ tien fibrin. vitA ch tkc dunfr hoal hoa nlnctninnffpn I t i ^ h
codon de bilu hien o P. pastoris, sau dd dugc lao d6ng trong vector pPICZoA va dua vAo h<ii nhlp vin bfl gene ci dflng tai tfl hgp dugc sing lpc dua vio kha nang bilu hi?n lumbrokinase. Protem tai to hgp dugc tinh sach qua cpt resin cfl gin nikel, kilm tra biing diSn di tren SDS-PAGE vi thir hoat tinh thiiy phSn tibrin. Kit qui cho thiy buoc diu da nhan dong va bilu hi?n thanh cflng lumbrokinase tii tfl hgp a P. pastons.
Tif khoa: H09I tinh thiiy phan fibrin, lumbrokinase, Pichia pastoris, tac nghen m^ch OAT VAN 0£
Lumbrokinase (LK) \d nhung chuoi pol^eptlde don gj^u asparagine hoac aspartic acid, rSt (t proline vd lysine, khong chi^a thSnh phan dirfrng. Chijng duvc xkp vdo logi senne protease kilm gi6ng trypsin. L4n d^u tiSn LK dirpc tdch chiet va d^t tSn Id lumbrokinase biri Mihara (Mihara, et al, 1989). DSy Id m$t nh6m enzyme c6 ho^t tinh thuy phSn fibrin cao, gom 6 isozyme (Mihara, etal.. 1991) vdi Khfii lu'fj'ng phSn tO •-25-40 kDa. Mgtsfi nghien CLFU chl ra rSng LKc6theh6a tan cgc mdu ddng vd h^n che kha nang vdn cgc, do dd co t h i dung dk dilu tri t i c nghen mgch mdu va b^nh tim mgch (Jin, ef al., 2000, Dong, et al.. 2004). Hi^n nay, LK va cac san phim c6 chirc nang tirong h/ da du^c thu'crng m^i hfla todn cku nhung cdc sdn ph^m deu dirgrc tdch chilt tir bdt giun thd. Tai Vt$t Nam, chua cd nghien CLPU ndo v l sdn mit LK tdi t6 hpp dung Idm thulc phdng ching tic nghen mgch mau vd nhO'ng nguyen iif u Idm ^ u l c phin iim deu phdi nh$p khiu. Vi v^y chung tdi d l xuit nghiSn ci>u xdy dg-ng qui trinh cdng ngh$ t^o cdc ddng te bdo san xuit LK tdi t l hcrp, Idm co sd d l sdn xuat thulc trong nirdc.
Trong cdc h$ bilu hl#n dirge sd dgng hi^n nay, nim men Pichia pastoris dirge si> dgng rdng rat nhlt d l bieu hi^n cdc protein eukaryote. Vi Id mdt eukaryote ndn cdc protein dirge bieu hi#n d t l bdo ndy thucmg dirge glycosyl hda vd cuOn xoln chtnh xac. Hon nOa, P. pastoris d l nuoi cay va cdc thao tdc di truyin don gian va chlJng cd t h i sinh tnrdng din m^t dd t l bdo cao. Ngoai ra, vector bilu hidn mang gene ngo^i iai c6 thi dirge ehdn hidu qua vdo genome nim men thdng qua qud trlnh tdi to hgp gli>a cdc dogn tirong ding tao ra ddng te bdo on djnh d l sdn sdng cho phep bilu hi$n protein ngogi lai. Vdl cdc tru dilm nli bdt, hg thong bilu hidn P. pastoris da dirge chiing tdi chpn Ig'a d l nghien ciru bleu hi$n LK, tgo nln tdng cho viec xay dgng quy trinh cdng nghd I n djnh sdn xuit LK tai to hgp. Trong hg thong bilu hi^n ndy, LK tdi t l hgp dugc bllu hidn vd tilt ra ngoai mOi tm-dng. Nhd vdy, LK thu nhdn dirge se cr dgng tan, ft tgp nhi&n, c6 clu true gan vdi tg nhidn vd do dd khdng bj mit hogt tlnh.
VAT LIEU VA PHlfONG PHAP V|t li^u
Chiing t l bdo E. coh DHIOB [F endA1 recAl galEIS gaLKLKTS nupG rpsL AlacX74 0BOIacZaM15 araOISS A(ara.leu)7697 mcrA &(mrr-hsdRMS-mcrBC) A ] vd chung nam men P. pastoris X33, co kieu hinh Mut' c6 nguon glc tir Invitrogen.
Plasmid pUC57 mang gene Ik nhdn du^c tir cdng ty Genescript (My). Vector nhan ddng pJet 1.2biunt vd vector bllu hi§n pPICZaA du'gc mua td Invitrogen.
LB (0.5% cao nam men, 1% NaCI vd 1% peptone), LB - amp (mdi tm-dng LB b l sung 100 pg/ml ampicilin), LB iowsait- Zeodn (mdi tnfdng LB chira 0.5% NaCi, 25 pg/ml zeocin), YPD (1% eao nam men, 2% peptone, 2% glucose/dextrose), YPD - Zeocin (mdi tm-dnp YPD b l sung zeocin pg/ml 100). YPDS- agar (mdi tnjdng YPD b l sung IM Sorbitol vd 2%
agar), BMGY (1% cao nam men. 2% peptone, 1.34% YNB, lOOmM potassium phosphate pH 6.0, 4.10"* % biotin, 1%
glycerol), BMMY (1% cao nam men, 2% peptone, 1.34% YNB, lOOmM potassium phosphate pH 6.0, 4.10"^ % biotin, 0 5% methanol)
C$p mil dge hidu nhdn gene Ik: pPICZoptLKmF (5'-CGGGAATTCATTGTTGGTGGTATTG-3') vd pPiCZoptLKR (5'- CGGTCTAGAGGGTTGTTAGTAATGATG-3')
Cdp mil AOXI: 5'AOXI (5'-gac tgg tte caa Itg aca agc-3') va 3'A0X1 (5'- gga tgt cag aat gcc att tge-3') Phuxj^g phap
Phan &ng PCR nhdn gene Ik vd tao dong ti bdo E. coli DHIOB mang vector tdch dong pjetl.2- LK (pJLK)
C CONG NGHE SINH HQC TOAN QUOC 2013
^ • ^ ^ K ^ ^ " ^ * " * ^ ^ ^^°^ ^ GenescripL My) dugc tien hanh PCR khuich dgi dogn gene ma hda LK vdi c$p mOi ^ hi$u LK chda vi t i cSt cOa 2 enzyme gidi hgn EcoRI va Xbal. Chu trinh phdn dng PCR: gS^C/S phOt; 30 chu 1^
(95 C/1 phut; 50'^C/45 gidy; 72°C/1 phut); 72°C/10 phut.
Sdn phlni PCR dugc n i i gh6p vdo vector pJet 1.2 blunt bing T4 DNA ligase theo chl din ctia hdng. Hdn hgp phan ung ndigh^) dugc ii vdi t l bdo khS bien E. coli DHIOB (ffi xu ly bdng CaCy trong 30 phiit vd soc nhiet 45 gidy a 42°C.
Dich blin i^p duvc b l sung 250 jd LB ldng, lac 1 gid d 37°C, 200 vdng/phut, hin hgp dugc trai trdn dTa mdi trudng LB '^^' " i M o J ^ " ^ ^'^ ' ^^ ^^- ^ ^ " "^^ '''^" "^P- ' ^ 1 ^ " ^ " ^ ^ " ^ sang Ipc va kiem tra, so sdnh vdi dli ehung Id vacior pjeti .2 khdng mang gene Ik d l thu nhgn ddng te bdo £ coff DH10B mang plasmid tdi to hgp.
Ueh chiit plasmid vd kiim tra bi„g enzyme dt giai h^n
Cdc khuan l9c dugc nhgt vd nudi lac 200 vdng/phiit d 37'C trwig mdi trudng LB - amp ldng td 14 -16 gid. Plasmid iWgc laeii chiettheo (rtiucmg phdp cua Brinboim vd Doly (1979), (Bimbotm & Doty. 1979). Nhimg plasmid cd kich thudc Wn hOTi p l a s m i d ^ chmig sS du^c chpn Ipe cho nhihig thi nghidm tilp theo. Do gene vd vector pJet 1.2 blunt dugc d t bang ^ enzyme Xba\ vd Xhoi tgo diu bing tnrirc khi nil ghdp tgo dix\g. nen su dung 2 enzyme d l kilm tra sg eo m$t cua gene/fr trong vector tach ddng pJet 1.2 Wunt ndy.
T90 dimg ti bdo E.coli DHIOB mang vector biiu Men pPICZaA - LK (pPLIQ
Plasmid pJLK vd vector bieu hidn pPICZaA dugc cdt bang enzyme gldi hgn EcoRI va Xbal d l thu gene Ik vd vector md vdng. D|ch cat sau fld dirge chgy dign di trfin gel 0,8% agarose vd duw: tinh sach bdng kit Aganase Gel Purification (Bioneer) theo chi dan cOa hang.
Dogn gene Ik vd vector pPICZoA md vdno sau khi tinh sach diroc nli ghep vdi nhau nhd T4 ligase d 22°C, qua dfim.
Plasmid dugc bien ngp vao t l bdo khd biln E. coli DHIOB theo phuang phdp sic nhidt vd sdng Ipe trdn mdi trudng LB low salt cd bd sung 25 pg/ml zeocin. D l chic chin cac plasmkl tai t l hgp da dugc chdn gene Ik, (^ting tw tiln hdnh cit kilm tra vdi EcoRI vd Xbai. sau dd didn di kilm Ira trdn gel aganase 0,8%.
Plasmid tdi t l hgp sau khi Wlm tra va «nh sgch bing each chilt vdi Chtorofomi: Isoamyl (24:1). dugc gdi dpc trlnh tg t^i cdng ty Macrogen (Hdn Qudc). Trinh tg dugc phdn tich trdn phin mim DNA Star.
7*90 dottg ti trdo P. Pastoris X33 mang gene Ik
Plasmid tdi t l hgp pPLK dugc c i t md vdng vdi enzyme Sad (thilt k l d l chdn vdo geneome nim men tgi vimg 5'AOX)- Mdt phan djch cat sau dd dugc chay di$n di trdn gel 0.8% agarose d l kilm tra vide d t md vdng hodn todn plasmid tdi t l hgp. Plasmid md vdng dugc tinh sgch bing kit (reaction cleanup, Qiagen) theo chl din cua hang.
Sdn phim d t plasmid tdi t i hgp sau tinh sgch duge bien ngp vdo nim men P. pastoris X33 bang phuang phdp xung didn. Djch biln nap sau dd fltfgc trai trdn dia mdi tardng YPGS (Yeast pepbsne glucose sorbitol) d$c cd b l sung 100 pg/ml zeocin fli chpn Ipc, flia duge u d SO^C trong 3 - 5 ngdy cho flin khi xuit hidn khuin Igc. Tiln hdnh ding thdi vdi 1 mlu doi chirng dm vdi t l bdo X33 biln ngp chda vector bilu hidn pPICZoA khing mang gene Ik. Khuin Igc flugc nudi d y d l thu nh|n bd gene vd PCR vdi cdp moi AOXI de kilm tra kilu gene. Chung tdi t i hgp sau khi dugc xdc nhdn mang gene Ik dugc i5$t tdn Id P. pastoris X33I pPICZoA- LK.
Kiim tra S{F biiu hl^n LK cOa chOng P. pastarts X33/pPICZaA - LK.
Chiing tdi t i hgp dugc hogt hda vd tdng sinh trong 10 ml mdi trudng BMGY b l sung 100 pg/ ml zeocin. lie 200 v&ng/phiit d 30°C trong 14 - 16 gid. Khi OD soo dgt tCr 2 - 6. thu sinh khli vd chuyin sang 25 ml mdi trudng BMMY, nudi lac 200 vdng/ phiit d 30°C. Cdm irng bing methanol ning d | culi 0,5% sau mil 24 gid nuii c l y Sau 96 gid d m dng, lytdm thu dich nudi d y va fli$n di trdn SDS- PAGE fli kilm tra sg bilu hidn LK.
Tlnh s^ch LK
Protein tdi t l hgp LK dugc tinh sgch qua cpt resin cd gdn nikel, kiem tra bang didn di trdn SDS-PAGE vd thd hoat tlnh thiiy phan fibin tren flia fibrin theo phuang phap cua Astrup vd Mullertz (1951) (Astrup& Mullertz, 1952).
KtT QUA VA T H A O LUAN
7(10 ddng ti bdo E. coli DHIOB mang plasmid til ti hgp pJet 1.2 - LK
Kit qua nhdn gene Ik td vector pUC57LK thu flugc 1 bang kfch thudc khoang 700 bp, phil hgp vdi kich thudc fy thuylt (Hlnh 1). Sdn phim ndy sau dd dupc nii ghdp vdo plasmid pJet 1.2 blunt nhd T4 ligase theo chl din cOa hdng. Hin hgp sau phdn dng nii ghep flugc biln nap vdo t l bdo E. cdi DH10B vd dugc sdng loc sa bp trdn mdi tnfdng LB - amp.
Cdc khuin Ige rieng rS vd ding nhlt dugc chpn d l kiem tra bdng each tach chilt plasmid vd flidn di kilm tra cd so sdnh kich thudc vdi doi chiing Id plasmid hfong dng khdng mang gene Ik. Sau do, cdc plasmid flugc chpn Ipc sa bd trdn sg dugc d t kilm tra vdl 2 enzyme gidi hgn id Xbal vd Xbol de kilm tra sg cd mdt cua gene Ik. Ket qud didn dl tren gei agarose 0.8% cho thiy, plasmid mang gene Ik cd kfch thudc cao han so vdi d i l chiing (Hinh 2) vd sau khi d t bdng enzyme gidi hgn da xuit hidn 1 bdng Id vector pJet 1.2 blunt (~ 3 ld>) vd 1 bang Id gene Ik (~ 750 bp), (Hinh 3). dieu ndy phll hgp vdi tfnh todn ly thuylt.
HOI NGHI KHOA HOC CONG NGHE SINH HQC TOAI.
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Hinh 1. PCR nli9n gene LK M, thang DNA Ikb.
2.1 vd 2.2. sfin phim PCR gene Ik. DCA. doi chirng dm
Hlnh 2. Kilm tra plasmid tai to hgp Hlnh 3. Kilm tra plasmid tii t l hgp pJet1.2-LK. 1-5, plasmid pJet 1.2 mang pJLK bang enzyme cit gW h^n. U, genefft.OC. plasmid pJet 1.2 thang Dh4A 1 kb. 1. plasmid tdi t l hgp pJLK d t bing 2 enzyme gidi ban Xhoi vd Xbal
Tao ddng ti bdo E.coli DHIOB mang vector biiu hien pPICZaA - LK (pPLiq
Sdn phim pJLK vd pPtCZoA d t vdi e n z ^ e gidi hgn EcoRI vd Xbal dugc tinh sgeh vd di^n di kiem tra trSn gel agarose 0.8%. Ket qud cho thiy cdc bdng khd sgch, diing kich thudc. du tidu chuan d l tien hanh cdc thf nghigm tilp theo (Hlnh 4). Cdc khuan Ige rieng re vd fling nhlt dugc chpn d l kilm tra bang cdch tach ehiet plasmid va dipn di kiem tra cd so sdnh kich thude vdi w i chimg Id plasmkl tuang ipng khdng mang gene Ik. Sau fld, cdc plasmid flugc chpn lpc sa bg trdn sd duge d t kilm tra vdi 2 enzyme gidi hgn Id EcoRI vd Xbal fle kiem tra sg cd mgt ciia gene Ik. Kit qua didn di trdn gei agarose 0.6% cho thiy, plasmid mang gene Ik cd kich thudc cao han so vdi floi chung (Hlnh 5) va sau khi d t bing enzyme gidi hgn dd xuit hidn 1 bdng Id vector pPICZoA (~ 3.6 kb) vd 1 bang Id gene Ik (- 750 bp) (Hlnh 6). phCi hgp vdi tlnh todn ly thuylt.
Hlnh 4. Tlnh s^ch sdn pham cat Hinh 5. Kilm tra plasmid tai t l hgp Hlnh 6 Kiem tra plasmid tdi t l hgp plasmid pJLKv&pPICZoAvdi 2 enzyme pPICZa- LK. DCA, plasmid pPICZoA. pPLK bang enzyme d l gldi hgn. M, gIM h^n EcoRI vd Xt»»l. M. thang DNA 1 1.2,4. plasmid pPICZoA khflng mang gene thang DNA 1 kb 1. friasmid tdi t6 hpp kb. 1,2, gene /k. 3, vector bieu hi§n Ik. 3, plasmid pPICZoA mang gen Ik pPLK d t bing 2 enzyme gidi hgn EcoFW pPICZoA (pPLK) vd Xbal.
Vector mang gene Ik dugc gidi trinh tu vd kit qud da khang djnh trlnh tg gene Ik Id chfnh xdc, gene dugc dua vdo vector bilu hidn diing theo thiet k l vd dung khung flpc md, dC dilu ki#n d l tiln hdnh tao chiing nam men tai t i hgp P.
pastoris X33 mang gene Ik.
T90 ddng ti bdo P. pastoris X33 mang gene Ik
Kit qud d t md vdng plasmid tdi t l hgp pPLK bang enzyme Sad thu dugc 1 bang kfch thudc khoang 4.5 kb. dOng theo tinh todn ly thuylt (Hlnh 7). Sdn pham dugc tinh sgch vd dign di trdn gei agarose 0.8% cho thay bang thu dugc sgch, ro rdng, dii didu kidn d l tiln hdnh bien ngp vao nim men P. pastoris X33.
Nhdng ddng ed kit qua PCR ti* bg gene nim men thu duge 1 bang Id gene AOXI ciia nam men (khodng 2.2 kb) vd 1 bdng Id dogn AOXI ciia pPLK (khodng 1.4 kb) (Hinh 8) se duge chpn Igc fli tiln hanh bieu hi§n LK.
Kiim tra sir bleu hi$n LK cOa chiing P. pastoris X33/pPICZaA - LK.
Dich nli cdc chiing^R pastoris X33/pPICZaA - LK nudi trong mdi trudng BMMY edm img vdi methanol 0.5% sau mSi 24 gid flugc dign di kiem tra SDS- PAGE cho thiy xuat hidn 1 bang kich thudc khoang 38 kDa, bang ndy khdng xuat hidn is mdu ddl Chung Id chung R pastoris X33/ pPICZoA Kfch thudc ndy cd the la do protein LK bj glycosyl hda khi bilu fii?n trong Chung nim men P. pastoris X33. Kit qua nay phii hgp vdl nghidn cdu eua Yan Hu vd cpng sg (Hu et al. 200a khi tidn hdnh bieu hi§n LK tir Lumbricus mbellus trong nim men P pastoris X33 nhd sir dgng vector bilu hidn pPICftiAi
vd thu dugc 1 bdng kfc^ thudc 38.61 kDa. ^
, C CONG N G H £ SINH HQC TOAN Q U 6 C 2013
fcf * i
. 2.2 kb
' 1-4 kb ^ ^ -^- 38 kDa
Hinh 7. K i l q u ^ « n h HJnh 8. K i t qua PCR b $ gene Hinh 9. K i t qud tinh sach Hlnh 10. Ket qua thir hogt tinh 59ch vector pPLK d i d t n i m men P. pastoris X33~
m d vong bang e n z p n e c | p m i l 5'AOXI vd 3'AOXI Sad. 1. plasmid pPLK. M, 1,2, sdn p h i m PCR M Ihang
»iang DNA 1 kb DNA 1 kb
protein tai t o hgp qua c$t thdy p h i n fibrin. 1, djch n i l resin g i n nikel. M. thang chuin pPLK/X33. 2, 3, 4. cdc phdn protein 14.4- 110 kDa. 1, dich n ^ dogn 2, 3, 4 qua cdt resin g i n PPLK/X33. 2, d|ch nfii nikel 5, d|ch enzym LK t i / pPICZaLK/X33. 3, 4, p h i n do^n nhien.
3,4 hnh sgch qua c^t resin.
Protein tdi ^ h g p c d khd n a n g b i l u hidn LK flugc tlnh s g c h qua cdt resin g a n nikel. S a n p h i m cdc phan doan flugc dien dl kldm tra S D S - P A G E . N h d n t h i y thu dugc 1 bang prctein sgch, kfch t h u d e khodng 38 kDa (Hlnh 9). D o n g t h d i , dich ndi t r u d e khi t m h s g c h v d edc p h d n d o g n d u g c t h d hogt tinh tren dTa fibrin nhdn tgo. K i t q u d b u d c fliu cho t h i y cde phdn d o g n d i u c d hogt tfnh t h i i y p h d n fibrin ( H i n h 10).
K £ T L U A N
Nghien c u u da tao d d n g v d bieu hi§n thdnh c d n g Lumbrokinase tdi t i h g p trong n i m men P pastoris X33. Tuy nhidn.
hogt l i n h c d n e h u a cao vi v d y n g h i d n c d u ndy id c o s d d l t i l p tue t l i u u nham tim ra d i l u kign thu d u g c LK tdi t i h g p c d hogt tfnh c a o n h l t , tgo tien fli cho vide nghidn c d u sdn x u i t lumbrokinase tdi t i h g p cho m i j c dfch d l l u trj t i c nghen mgch m d u .
L^McamffD
Nghien cdu nam trong khuon kho dS ldi Nghiin cihi guy trinh cong nghf sdn zudi Lumbrokinase Idi Id hpp ldm thuoc phong chong lie nghen mgch mdu ", mdsoKC 04/11-15, diudc Knh wc ky Ihugl vd cdng ngh$ Xin dupc gui lai cdm an lin Yi^n Cong nghe smhhoc- Vifn Mdn Idm Khoa hoc vd Cong nghf^ Vi4t Nam. HQC vi$n Qudn y- 8$ quae phong, Khoa huyel hpc- Bfnh vifn Trung uang Qudn Opi 108 vd Vifn Tlm mach- Bfnh vifn Bgch mai da phoi Aop, lao dieu hen vd giup da chiing loi Ihifc kifn di lai ndy.
T A I L I E U T H A M K H A O
/^stmp T & Mullertz S (1952). The fibrin plate melhod for estimating fitmnolytic activity. Amh Bkichem Biophys 40:346-351.
Dong GQ, Yuan XL, Shan YJ. Zhao ZH, Chen JP & Cong YW (2004). Molecular cloning and characterization of cDNA encoding fibrinolytic enzyme-3 from earthwomi Eisenia foetida. Acta Biochim Biophys Sin (Shanghai) 36' 303-308.
Hu Y, Meng XL. Xu JP. Lu W & Wang J (2005). Cloning and expression of earthvrami fibrinolytic enzyme PM(246) in Pichia pastoris.
Pmtein Expr Purif 43:18-25.
Jin L, Jin H. Zhang G & Xu G (2000). Changes in coagulation and tissue plasminogene acHvator after the treatment of cerebral inbrction with lumbrokinase. Clin Hemoriieol Microcirc 23' 213-218.
Mihara H, Yineta T, Sumi H, Soeda M & Manjyama M (' Thrombosis Haemost 62: 545-549.
) A possibility of earth worm powder as therapeutc agenet for thrombosis Mihara H, Sumi H, Yoneta T Mizumolo H, Ikeda R. Seiki M & Manjyama M (1991). A novel fibnnolytic enzyme extracted from the earthwomi, Lumbricus rubellus Jpn J Physiol 41: 461-472
CLONING AND EXPRESSION OF LUMBROKINASE IN PICHIA PASTORIS
Vu T h i B i c h N g o c , L y T h i B i c h T h u y , Q u y e n D i n h T h i
Institute of Biotechnology. Vietnam Academy of Science and Technology
S U M M A R Y
Thrombosis, the blockage o f blood vessels with clots, can lead to acute myocardial infarction and ischemic stroke, both lead to death.
For acute cases, treatment may be surgical interventions to remove blood clot or bypass the blockage. However, chronic or risk cases
HOI NGH! KHOA HOC C O N G N G H E SINH HQC TOAw •
should take long — term drug treatment to prevent blockage Lumbrokinase, a group of fitninolytic enzymes from earthworm, have the molecular weights of 25 to 40 kDa. These enzymes can not only hydrolyze fibrin diiecdy but also activate plasminogen to plasmin - the active fitmnolyiic enzyme of 4 e body. Therefore, lumbrokinase now has been widdy used ftir the treatment of*
thrombosis. In this study, the genee encodmg lumbrokinase ftom the earthworm Lumbricus rubellus (GeneBank Accession No.
AF304199) was codon- optimized for the expression in P. pastoris, sul>-cloned into pPICZoA vector and tbcn integrated into the geneome of host cell. The recombinant clones were screened twsed on the ability of lumbrokinase expression. The recombinant protein was purified by Ni-NTA His'Bind Resin, checked by SDS-PAGE and fibrinolytic activity assays. Tlie results showed the primarily success in the cloning and expression of recombinant lumbrokinase m P. pastoris.
Keywords: Fibrinolytic activity, lumbrokmase, Pichia pastoris, lfaromt>osis.
' Author for corresspondence: Tel: +84-4-37568260; Email quyen@ibt ac vn