B Y T TRNG I HC DC H NI L TH TNH CHIT TCH V XC NH HOT TNH CA RIBONUCLEASE T TRNG CC (Bufo sp., Bufonidae) V TRNG CH (Rana rugolusa., Ranidae)

KHO LUN TT NGHIP DC S H NI 2014

B Y T TRNG I HC DC H NI L TH TNH CHIT TCH V XC NH HOT TNH CA RIBONUCLEASE T TRNG CC (Bufo sp., Bufonidae) V TRNG CH (Rana rugolusa., Ranidae)

KHO LUN TT NGHIP DC S Ngi hng dn: TS. Nguyn Vn R Ni thc hin : B mn Ha Sinh H NI 2014 LI CM N Tixinbytlngbitnchnthnhvsusctithygio,TS. Nguyn Vn R bmn Ho Sinh, Trng i hc Dc H Ni tn tnh hng dn v gip ti hon thnh kho lun ny. Ti xin chn thnh cm n s gip ca anh Nguyn Quc Ton cng ti hon thnh ti ny,cbit ls gip to iu kincacc thyc, ccch kthut vinca bmnHo Sinh, Trng i hc Dc H Ni. Ti cng xin trn trng cm n Ban GimHiu, Phng o To, cc phng ban, thy c gio Trng i hc Dc H Ni to iu kin thun li cho ti trong sut qu trnh hc tp ti nh trng. Cui cng, ti xin by t lng bit n chn thnh ti gia nh, bn b ng vin, gip ti hon thnh nhim v ca mnh. H Ni, ngy 14 thng 5 nm 2014 Sinh vin L Th Tnh MC LC T VN ............................................................................................... 1 CHNG 1: TNG QUAN ........................................................................ 2 1.1. C IM V TRNG NG VT LNG C ............................................. 2 1.2. C IM CHUNG V RNASE T TRNG NG VT LNG C .......... 3 1.2.1. Mt s nghin cu RNase t trng ng vt lng c ....................................................3 1.2.2. Cu trc phn t ca RNase t trng ng vt lng c ................................................5 1.2.3. C ch xc tc ca RNase t trng ng vt lng c ...................................................6 1.2.4. Hot tnh gy c t bo ca RNase t trng ch............................................................8 1.2.5. Cc yu t nh hng n hot ng ca RNase ............................................................9 1.3. CC PHNG PHP CHIT TCH V XC NH HOT RNASE ........12 1.3.1. Cc phng php chit tch enzym RNase .................................................................. 12 1.3.2. Cc phng php xc nh hot ca RNase ............................................................ 14 1.4. KH NNG NG DNG CA RNASE ............................................................14 2.1. NGUYN VT LIU V THIT B ...................................................................16 2.1.1. Nguyn liu .................................................................................................................. 16 2.1.2. Ho cht ........................................................................................................................ 16 2.1.3. Thit b .......................................................................................................................... 16 2.2.2. K thut kt ta phn on protein - enzym ................................................................. 17 2.2.3. Phng php xc nh hm lng protein .................................................................... 19 2.2.4. Phng php xc nh hot RNase .......................................................................... 22 3.1. KT QU CHIT TCH RNASE T TRNG CH V TRNG CC ............24 3.1.1. Chit RNase t trng ch ............................................................................................. 24 3.1.3. Chit RNase t trng cc ............................................................................................. 24 3.1.4.Nhnccphnonproteint dchchitmtrngccbngkthuttaphnon vi (NH4)2SO4 ......................................................................................................................... 25 3.3.1. Kt qu xc nh s phn b hot RNase t trng ch ...................................26 KT LUN V XUT ......................................................................... 35 Kt lun .......................................................................................................................35 xut ........................................................................................................................35 DANH MC CC K HIU V CC CH VIT TT ADN Axid Deoxiribonucleic ARN Axid ribonucleic Amph Amphinase BS RNaseBovine seminal ribonuclease Cpr Nng protein DCDch chit ddDung dch vE n v enzym HRHot enzym Ribonuclease HRring Hot ring ca enzym Ribonuclease MW Khi lng phn t ODMt quang hc ONC Onconase PCR Polymerase Chain Reaction PBSPhosphate buffered saline pHopt pH ti u PhPhn on PrProtein RNase Ribonuclease TC Trng cc TE Trng ch TT Thuc th TTH Trung tm hot ng

DANH MC CC BNG Bng2.1Tlphaccdungdchxydngngchun protein19 Bng2.2Gitrmtquangcadungdchproteinbitnng ...21 Bng3.1Hmlngproteintccphnondchchittrng ch.25 Bng3.2Hmlngproteintccphnondchchittrng cc.26 Bng 3.3Ktquxcnhhot RNasetrong ccphnondchchit proteinnhnct DCTEa.........27 Bng 3.4Ktquxcnhhot RNasetrong ccphnondchchit proteinnhnct DCTEb.....28 Bng 3.5Ktquxcnhhot RNasetrong ccphnondchchit proteinnhnct DCTCa.................29 Bng 3.6Ktquxcnhhot RNasetrong ccphnondchchit proteinnhnctDCTCb.......30 DANH MC CC HNH V, TH Hnh 1.1 Trng ng vt lng c ........2 Hnh 1.2 Cu trc khng gian ca RNase A .... ....5 Hnh 1.3 nh chp 2AAS-NMR 3D ca RNase ....6 Hnh1.4CchphnngthuphnRNAcaRNasetheo2giaion [17].7 Hnh 1.5 C ch xc tc kim acid c s tham gia ca His 12 v His 119 [26]....7 Hnh 1.6 Hot tnh cytotoxin ca RNase [26]..8 Hnh 1.7 Cu trc ba chiu ca RI (mu ) v RNase A (mu xanh) [2611 Hnh2.1SccgiaionchittchRNasettrngchvtrng cc................................................................................................................18 Hnh2.2thtngquangianngproteinvhpth quang..21 1 T VN Ung th l mt trong nhng nguyn nhn gy t vong hng u trn th gii c bit l cc nc ang pht trin. Nm 2012, c 8,2 triu ngi trn th giicht v ung th.Vichi ph iu trcao, hiu quiu tr thp, ung thtrthnhgnhnngchobnhnhn,gianhvxhi.Sphttrin khngngngcakhoahcemnchonhn loinhng thnhtuvt bc,gipchtlngcucsngngymtnngcao. Tronglnhvciutr ung th cn bnh vn c coi l nan y cng ang c nhng bc t ph em li cho ngi bnh nhng hy vng mi. Gn y, cc nh nghin cu thuc trng i hc Bath v tng cng tyAlfacellphnlpthnhcngenzymOnconasevAmphinasethuc nhmRibonucleasettbotrngch(loiRanapipiens).Ccenzymny v ang c tinhnh th nghimtin lmsngv lmsng trongiu tr ungthcbitlungthno.TiVitNam,mtsbi thucngy dngccchabnhungth[1].Trongkhi,ccvch u thuclp ngvtlngcsinhsngnhiuvngtrongcnctonnngun nguyn liu di do cho nghin cu enzym. Tuy nhin, nc ta cha c cc nghin cu su mc phn t v Ribonuclease t trng ch v trng cc. Do,chngtitinhnhti:Chittchvxcnhhottnhca Ribonucleasettrngcc(Bufosp.,Bufonidae)vtrngch(Rana rugolusa., Ranidae) vi cc mc ch sau: - Chit tch c ch phm RNase t trng cc (Bufo sp., Bufonidae) v trng ch (Rana rugolusa., Ranidae) c mc tinh sch khc nhau. - Xc nh c hot RNase trong cc phn on dch chit ca 2 loi trng cc v trng ch. 2 CHNG 1: TNG QUAN 1.1. C IM V TRNG NG VT LNG C ch v cc phn b nhiu ni trn th gii. Vit Nam, chng sinh sng nhiu vng to nn ngun nguyn liu phong ph cho nghin cu. Trngngvaitrquantrngtrongsinhsn,chabnhimscth mangthngtinditruyn.Bivy,trongtbotrngc nhiuloienzym- proteinthamgia vocc qutrnh chuyn ha, sao chp,phinm,dchm v tng hp protein duy tr b gen ca loi. Enzym RNase t t bo trng ch c c bit ch trong thi gian gn y vi hot tnh gy c t bo (hot tnh cytotoxin) c chit tch v tin hnh cc th nghim hng ti iu tr ung th. ch v cc thuc lp ng vt lng c. Do , cu to ca trng u c cc c im chung ca lp lng c gm nm phn nh hnh v sau: Hnh 1.1 Trng ng vt lng c 1. Lp v nhy 4. Nhn trng 2. Mng Vitellin 5. Phi 3. Cht lng Perivitellin3 Lp v nhy bo v phi khi tc ng t mi trng bn ngoi. Mng vitellinbaoquanhtrngcbnchtprotein.Khnggiangiamngvitellin v phi c cha y cht lng perivitellin. Nhn trng nui dng phi.TbotrngcangvtlngccenzymRNasevnhiuloi enzym khc phc v cho cha nng sinh sn v dinh dng ca trng. 1.2. C IM CHUNG V RNASE T TRNG NG VT LNG C RNase thuc nhm enzym thy phn [9], c c cht c hiu l ARN. Hin nay, trn th gii pht hin hn 100 thnh vin thuc siu h RNase Av9loiRNasengi[19],[21],[22],[23],[38],[43],[45].Ccnh khoa hc phn lp c RNase t t bo trng ch (Rana pipiens) c hot tnh gy c t bo [14], [17],[33]. 1.2.1. Mt s nghin cu RNase t trng ng vt lng c T nhng nm 1960 1970, RNase A, EC 3.1.27.5 tr thnh m hnh mutrongcccngtrnhnghincuenzymhcnhuthvnguntip cn,khnngtinhchddngcngnhkchthcphntnh~14kDa. RNase A l enzym u tin c xc nh trnh t acid amin v l enzym th bacxcnhcutrcsauLysozymevCarboxypeptidaseA[17],[26], [37]. Onconase v Amphinase l hai RNase lng c u tin c phn lp v xc nh trnh t bi tng cng ty Alfacell c hot tnh gy c t bo[51]. Onconase (ONC) cn gi l Ranpirnase c chit tch t t bo trngch loi Rana pipiens vi hot tnh gy c t bo (hot tnh cytotoxin) [11], [27], [29], [44]. Onconase ch yu tn cng vo giai on G1 c ch chu k t bo [12]. TngcngtyAlfacelltinhnhthnghimlmsnggiaionIRanpirnase bnh nhn u trung biu m c tnh v ung th phi khng phi 4 t bo nh. Hon thnh giai on III th nghim n c Ranpirnase bnh nhnutrungbiumctnhvothngT nm1999[32]. GiaionIIth nghimlmsng Ranpirnasetrongiutr ungth v v ungth thc qun honthnhvonm2006[31].Alfacellangtiptcthnghimlmsng giaionIIIbRanpirnasecngvidoxorubicin[29],[35].Ccthnghim cho thy nhiu dng t bo ung th nhy cm vi Onconase [30]. Alfacell nhncchnbngsngchMvbnbngsngchchuuv ranphirnase. AmphinaselenzymthhaiccngtyAlfacellphnlpvth nghimthnhcngsauOnconase,mtphinbncaenzymONCttbo trng ch Northern Leopard (Rana pipiens) c trin vng rt ln trong iu tr ungth,cbitlungthno.Liuphpminyangcthnghim tin lm sng [14], [40]. 5 1.2.2. Cu trc phn t ca RNase t trng ng vt lng cOnconase v amphinase thuc siu h RNase A c cu trc phn t gn tng ng vi RNase A. Hnh 1.2 Cu trc khng gian ca RNase A [48] RNase A c khi lng phn t nh ~14 kD vi 124 gc acid amin, 19 trntngs20loiacidamintrongproteincmtcutrcnytr Trytophan. Cng thc phn t dng khng tch in l C575H907N171O192S12 vi MW = 13 686 Da. V hnh dng, RNase A ging qu thn, cc gc ca trung tm hot ng nm trong vng hc - l vng lm ca qu thn. Cu trc bc hai in hnh ca RNase bao gm bn phin cu trc i song song ngc chiu k bn cnh ba chui xon ngn. Trong phn t c bn cu ni disunfid c hnh thnh t tm gc cystein cc v tr 26 84, 40 95, 58 110 v 65 72. Cc cu ni ny c th b ph v bi mercaptoethanol, to thnh tm nhm - SH t do lm phn t enzym b dui ra v mt hot tnh xc tc [37]. 6 Hnh 1.3 nh chp 2AAS-NMR 3D ca RNase Mu xanh l nhm amin, mu l nhm carboxyl, mu trng l vng trung tm hot ng. OnconasectinhschttrngchloiRanapipienschottnh chngungth.ProteinnycnckhiulP30[13],cutotmt chuipolypeptidvi104acidamin,MW=12000Da,imngin khong 9,5 [11], [51]. Onconase kh tng ng viRNase A3, ging nhau ti30% trnh t axid amin v tng t v cu trc khng gian 3 chiu. Amphinase (Amph) c bn bin th c phn lp, cha 114 acid amin trong chui polypeptid v c N-glycosyl ha hai v tr. Trnh t acid amintngnggiaccbinthl86,8-99,1%vgiaccbinthvi ONC l 38,2-40,0% [41]. 1.2.3. C ch xc tc ca RNase t trng ng vt lng c Ging nh RNasekhc,cchtcaRNasettrngch lARNvc tnh c hiu pyrimidin. N phn ct lin kt C5-O-P ng sau pyrimidin m khngphnctlinktC5-O-Pngsaupurintothnhdnxut 7 pyrimidin - 2,3 - phosphat vng. RNase cng xc tc thy phn to nn cc phosphat vng cytidin - 2,3- phosphat v uridin - 2,3- phosphat [18], [37]. PhnngsinhhadoRNasexctclphnngthuphnlinkt phosphodiestertrongphntacidnucleic.QutrnhthuphnARNdo RNase xc tc xy ra qua 2 giai on theo c ch xc tc kim acid vi s tham gia ca 2 gc His-12 v His-119: Hnh 1.4 C ch phn ng thu phn RNA ca RNase theo 2 giai on [17] Hnh 1.5 C ch xc tc kim acid c s tham gia ca His 12 v His 119 [26] 8 Giai on 1: Phn ng din ra nhanh v c th o ngc, phn ct chui polymer to ra sn phm trung gian l 2, 3 nucleotidm khng gii phng nhm acid. Trong giai on ny, His-12 ng vai tr l cht xc tc kim nhn proton H+ v tr 2' cn His-119 ng vai tr l acid proton ho nhm i khi RNA-O-. Giai on 2: Din ra chm hn v gii phng nhm acid th hai thuc gcphosphattrongqutrnhthuphndnxutphosphatvng.Tronggiai onnyvaitrca2gcHis-12vHis-119ngcli:His-119tngtc viH2OtheocchxctckimnhnprotoncnHis-12mnhnvic proton ho nhm i khi sn phm v tr C-2' [18], [37], [44]. 1.2.4. Hot tnh gy c t bo ca RNase t trng ch RNase ng vai tr quan trng trong nhiu qu trinh sinh hc. RNase 1 gip tiu ha thy phn ARN trong thc n [43]. RNase H tham gia vo qu trnhsinhtnghpANDvARN[46].BSRNasethamgiacchmin dch[24].CcRNasecngungcttbobchcuaacidchottnh khng khun [39], chng virus [38], chng k sinh trng [21] v tham gia vo h thng min dch ca c th [45]. a s cc RNase u b c ch bi RI, ch c mt s RNase c kh nng thot khi s c ch ca RI gy c t bo. Hnh 1.6 Hot tnh cytotoxin ca RNase [26] 9 Giai on I: RNase tng tc vi cc receptor trn b mt t bo. Giai on II: RNase xm nhp vo bn trong t bo. Giai on III: RNase thot khi RI v thy phn ARN. C s ca hot tnh cytotoxin l kh nng thot khi s kim sot ca proteincch(RI)trongbotng[20],[27],[37]nhRNaseddng phn ct c cht ARN c hiu trong t bo. Onconase khng b c ch bi RI c t bo cht. Onconase bm trn bmttbonhccth th,xmnhpvobn trongtbovthyphn ARNcatbo.Do,ONCcchqutrnhtnghpproteinvgy cht t bo [44]. Amphinaseckhnngnhnbitvgnvomtloingtrn mngtboungth,quaxmnhpcvobntrongtboungth, ph hy ARN, ngn cn qu trnh nhn ln ca t bo ung th [18].1.2.5. Cc yu t nh hng n hot ng ca RNase RNase l mt protein nn hot ng ca RNase cng ph thuc vo cc yu t nh hng n hot ng chung ca mt protein enzym. Nngccht:Nngcchttng,tc phnng xctcca enzymtng.Khinng cchttngnmccaothtcphnng t cc i. Thi im ny enzym bo ho c cht. C cht ARN d b thy phn cbittrongmitrngkim,gysailchtrongxcnhhotenzym [9]. Nng enzym: Tc phn ng xc tc enzym t l thun vi hm lngenzym.Khitngcaonngenzymthtcdphnngkhngtng 10 na do cc sn phm to ra nhiu s tc dng vo v tr d lp th ca enzym lm cho phn ng t bo ho [9]. TcdngcapH:CcnghincutrcyvRNasechothyhu ht cc enzym ny th hin hot tnh cao nht vng pH 6 n pH 9, pH thch hpnhtchoRNasehotnglt7n8.NupHqucaohayquthp hottnhRNaseubgimhaybinmt.Vd,RNasenmmenruc pHopt =7,6[18],[44].TuynhinpHopt caRNasetncrnhmangen Vit Nam c gi tr rt acid pHopt = 2,62 0,16 khc vi tt c cc RNase c bit n cho ti nay [8], [9]. Tcdngcanhit:Nhitngvaitrquantrngtrongphn ng thu phn ARN ca enzym. Hu ht cc RNase c nhit thch hp cao khong 40-700C. Mt s enzym rt bn vi nhit nhng hot tnh khng tng tuyn tnh vi nhit nh RNase nc rn h mang en ca Vit Nam: 25-490Chottnhenzymctngcng,t59-900Cthhotenzymgim ngkvti1000Cthhottnhlicaonht[7].Mtsenzymkhcth kmbnnhit,vd:RNasetrongncrnhmangvngTrungLinX (Naja oxiana) bmt 50% hot tnh khi un cch thu 5 pht 500C vmt hon ton hot tnh khi x l nh vy 700C [15], [42]. Tc dng ca ion kim loi: Tc dng ca ion kim loi rt phc tp v n tng tc vi TTH v c ch xc tc ca enzym [9]. V d Mg2+ nng 10mM lm tng hot tnh RNase trong rn h mang (Naja oxiana) ln n 10 ln [15] cn nng nh hn 2mM hu nh khng gy nh hng n hot tnh nhng nng 4mM li c ch 50% hot tnh ca RNase rn h mang en Vit Nam [7]. 11 Tc dng ca cht hot ho: Cc cht hot ho gip tng cng hot tnh thu phn ca RNase trong gii hn nng xc nh v thng c bn cht ho hc khc nhau: - Cc hp cht hu c nh 5,6-dimethylbenzimidazole, benzynimidazole, cc cht ty ra tch in dng cng c kh nng lm tng hot tnh RNase. - Cc acid amin t nhin c trong c th nh glycin, histidin cng hot ho RNase.CcchttrnhothoRNasetheonhngcchkhcnhaunhng cng thng nht kh nng kt hp trc tip vi phn t RNase, lm thay i cu trc khng gian ca n theo hng c li cho hot tnh xc tc hoc gip loi tr cc yu t km hm khi mi trng phn ng [43]. Tcdngcachtcch:VaitrcaccproteinkmhmRNase (RI)cnchacphthintonb.Ccnhnghincuaranhiu gi thit cho vai tr sinh hc ca RI: iu ho hot ng ca RNase, bo v t bo khi c tnh ca cc RNase, theo di trng thi oxy ho t bo vi cc nhn t nh tui tc, stress Hnh 1.7 Cu trc ba chiu ca RI (mu ) v RNase A (mu xanh) [26] 12 RIc khilng phnt khong50000Da,chim 0,01 0,1% tng s protein trong bo tng. Ring nhau thai v no t l ny ln hn, tng ng l 0,1% v 0,08% [20], [37]. Cu trc ba chiu c trng bi cc n v xonvsi.PhntRIcilcmnhviRNase,chngtophcvi nhiu thnh vin thuc siu h RNase A theo t l 1:1. Mi phn t RI cha t 30 32 gc Cys dng kh nnmi trng kh nh dch bo rt ph hp duytrhottnhRI.VicoxyhomtgcCysskotheosoxyhocc gccnli.Khiboxyho,RImtkhnnglinktviRNasevnhanh chng b thu phn bi protease trong t bo [16], [25]. Tuy nhin, kh nng bo v t bo ca RI cng b hn ch bi mt s enzym. Hu ht cc thnh vin thuc siu h RNase A c cu trc si n u b km hm bi RI c trong dch bo, ngoi tr ONC v Amph.1.3.CCPHNGPHPCHITTCHVXCNHHOT RNASE 1.3.1. Cc phng php chit tch enzym RNase RNaselmtproteindotacthpdngccphngphpchit tch protein. RNase trong t bo trng l enzym ni bo nn cn ph v t bo gii phngenzym.Ccphngphpcthdngphvtbonh:phng phpchc(nghin,dngsngsium),sdngchttyra,dng enzym[2], [4], [10]. Mt s phng php chit xut enzym- Phng php ta phn on: da trn kh nng ta ca enzym protein nhng nng mui khc nhau hoc ta trong mt s dung mi hu c nh alcol, ceton[6]. 13 Dngmuivc:datrnkhnngtakhcnhaucaproteincc nng bo ha khc nhau. Cc mui c th s dng nh: (NH4)2SO4, Na2SO4trong(NH4)2SO4 thngcsdngnhiunhtdo ha tan trong nc cao (720g/l 25o C), t c vi enzym trong nhiu trng hp cn gip n nh enzym m gi thnh li r [3], [5]. Dng dung mi hu c nhit thp: dung mi hu c c kh nng trn ln vi nc thm vo dch chit s lm thay i hng s in mi gim tancaenzym- protein to kt ta.Thng s dng:aceton, ethanol[2], [4]. -Phngphpgybintnhchnlc:datrnpH vnhit,thngp dngvienzymbnnhit hocbntrongmitrngacid.Theomts kt qu nghin cu, RNase kh bn nhit nn c th p dng c phng php ny. - Mt s phng php khc: c th s dng polymer [2], [36], kt ta enzym ti im ng in Mt s phng php tinh ch enzym - Phng php sc k:Scklcgel:davoskhcnhauvhnhdngvkchthcca enzym.ThngsdnggelSephadexvinhiuloikchthcmt likhcnhau.Phngphpnytinhnhnhanhcthtinhch enzym t ch phm th, khng cn th tch dch chit ln, kh ph hp trong nghin cu phng th nghim tuy nhin li lm long ch phm [3], [5], [36]. Scktraoiion:datrnphnngtraoiiongiaenzym- proteinvinhatraoiion.Phngphpnycthtchranhiu 14 phn on protein nhng i vi enzym cha bit cc tnh cht th vic lachnchtmangvdungdchphnhpphskhkhn[3],[5], [36].- Phng php kt tinh: thng s dng (NH4)2SO4. Phng php ny thc hin d dng tuy nhin enzym - protein thu c b ln mui do phi tin hnh loi mui bng cc phng php nh: thm tch, lc gel [5], [36]. - Cc phng phpkhc: phng php thm tch, phng php siu ly m, phng php in di 1.3.2. Cc phng php xc nh hot ca RNase V nguyn tc c hai phng php chnh sau:-olngcchtbmtihaylngsnphmctothnhtrongmt thi gian nht nh ng vi mt nng enzym xc nh. -othigiancnthitthucmtlngbinthinnhtnhcac cht hay sn phm tng ng vi mt nng enzym xc nh. C th xc nh hot cho mi enzym bng cch s dng c cht c hiu. RNase c c cht c hiu l ARN nn c th s dng phng php ny xc nh hot enzym. 1.4. KH NNG NG DNG CA RNASE Hinnay,RNasevangcngdngnhiutrongcngngh sinh hc. V d nh ch phm enzym NZY RNase H (E. coli) trn th trng csdngtrong:todngphntbngcchxctcctchiuARN ca th laiARN:ADNmkhngct ADNhoc ARNkhng trong th lai; dng ph hu khun mu ARN sau khi tng hp ADNC th nht; vic x ltrcADNvienzymgipcithinnhyvbovmilinktvi mi trong phn ng PCR 15 Khng ch dng li trong cng ngh sinh hc, vi vic phn lp v pht hinhottnhgyctbocaRNasettbotrngchtratrin vng ln trong iu tr ung th c bit l ung th no v cc loi ung th c tnh. Onconase c th nghim lm sng giai on I, giai on II v giai on III iu tr cc khi u rn, bao gm ung th phi v ung th tuyn ty [30],[31],[32],[34].Trongccthnghim,ONCctcngtchccn thigiansngtrungbnhcabnhnhn.Tuycctnhtrnthnliu thp,nhngcthongcsaukhingngiutr.Onconasecncch virus HIV type 1 ngi [46]. Amphinase cng ang c tin hnh cc th nghim tin lm sng. c bit,amphinasecbocockhnngtmkim,xmnhprtcaovo cc t bo ung th do nhn ra lp ng c th trn b mt t bo ung th v bmchtvo.Sau,Amphinasexmnhpvobntrongtbo,thy phn ARN gy c t bo [40]. Tnh chn lc v xm nhp vo bn trong t bo ung th cc v tr su m ra hng i mi trong iu tr ung th no mt loi ung th c bit n l kh iu tr. Nhng nm gn y, cc nh khoa hc cn tp trung sn xut ra cc ch phm RNase nhn to c hot tnh chng ung th nh vic vn dng cc k thut cng ngh sinh hc hin i mc phn t. Dng bin th ca RNase c to ra bng cch thay th mt hay vi gc acid amin cn cho tng tc protein enzym hoc to thm lin kt disunfid lm tng bn vng cu trc v gim bt s nhy cmvi cht c chhayccproteasetrongtbo.CcbinthcaRNasecngctora bng cch gn c hiu vo RNase nhng phn t protein lin kt c hiu vi nhng t bo nht nh cho php to ra nhng cht c chn lc hiu qu [28]. 16 CHNG II. NGUYN LIU V PHNG PHP NGHIN CU 2.1. NGUYN VT LIU V THIT B 2.1.1. Nguyn liu -Trngch(Rannarugulosa.,Ranidae)vtrngcc(Bufosp., Bufonidae) thu mua ch Vnh Tuy - H Ni, sau c mang v bo qun trong ngn t lnh. 2.1.2. Ho cht - ARN nm men mua ca hng Sigma - Dung dch m PBS pH = 7,4 - Dung dch acid sufuric 0,25 N - Dung dch casein 1% - Thuc th Gornal - Mui (NH4)2SO4 2.1.3. Thit b- Cn k thut TE412, Sartorius - Cn phn tch TE214S, Sartorius - My ly tm lnh Z 32 HK - My o pH Mettler Toledo -MyoquangphhpthSpectroUVVISDoubleBeamPC Scaning Spectrophotometer UVD 2960 - Dng c thu tinh kh v sch 17 2.2. CC PHNG PHP NGHIN CU 2.2.1. Phng php chit tch enzym Nguyn tc: Da vo kh nng ha tan enzymca dung dch H2SO4 0,25 N v dung dch m PBS pH 7,4 sau khi tin hnh ph v t bo. Dung dchH2SO4 0,25Nctcdngvhiuhoccenzymthuphnctrong lizosom t bo ng vt. Dung dch m PBS pH 7,4 c pH v thnh phn ion (cha K+, Na+) gn ging vi iu kin trong t bo. Tin hnh: Trng ch 25g hoc 10g trng cc c nghin nh trong ci vi 60 ml 80 ml dung dch H2SO4 0,25N hay dung dch m PBS pH 7,4 lnh v 18 20h ngn mt t lnh. Ngy hm sau, em ly tm vi tc 6000 vng trong 1h 40C loi cn v thu dch li tm, gi l dch chit m. 2.2.2. K thut kt ta phn on protein - enzym Nguyn tc: Da trn c s khc nhau v kh nng ta protein cc nngmuibohakhcnhau.Khithmmuivodungdchprotein enzym, tnh tan ca protein enzym thay i, nng mui thp tan tng nhvgimmnhnngmuicao.Sgimtancaproteinenzym dn ti kt ta. Tin hnh:T cc DC thu c trn, chia DC thnh bn phn bng nhau. Thm t t mui (NH4)2SO4 vo DC ln lt t bo ha 20%, 40%, 60%, 80%. Khuy u, kt ta qua m trong ngn mt t lnh. Ngy hm sau, em ly tm lnh 1h 40C thu ta. Ta thu c em ha tan li trong dung dch H2SO4 0,25N hay dung dch m PBS pH 7,4 lnh. 18 - Nghin nh trng trong ci . -ThmddPBSpH=7,4hocddH2SO4 0,25N 18 24h trong t lnh. - Ly tm lnh 4oC trong 1h, thu dch.

- Chia DC lm 4 phn bng nhau.-Thmmui(NH4)2SO4voDClnltt bo ha 20%, 40%, 60%, 80%, kt ta qua m. -Ly tm lnh 4oC trong 1h, thu ta.

- Ha tan vi PBS pH 7,4 hay H2SO4 0,25N. Hnh 2.1 S cc giai on chit tch RNase t trng ch v trng cc Phn on ta enzym protein Ph v t bo trng Thu trng Dch chit m 16 loi DC19 2.2.3. Phng php xc nh hm lng protein Cc phng php nh lng protein thng dng hin nay l Kieldahl, Biuret, Lowry, o quang trc tip v phng php cn [6]. Trong ti ny, tisdngkthutnhlngproteinbngphngphpBiuret(kthut Gornal). Nguyntc:Protein(clinktpeptip)khiphnngvithucth Gornal trong mi trng kim s cho phc hp mu tm hng. o quang ph hp th ca dung dch th, so vi biu mu nh lng protein. Tin hnh: Xy dng ng chun nh lng protein- Pha dung dch casein 1% - Pha dung dch m PBS pH = 7,4, pha thuc th Gornal Thc hin phn ng theo bng, lc u, cc ng 30 pht nhit phng sau em o mt quang bc sng 550 nm, cuvet 1cm. Bng 2.1 T l pha cc dung dch xy dng ng chun protein Thnh phn1234567891011 dd casein 1% (ml) 0,10,20,30,40,50,60,70,80,91,00 dd m (ml)0,90,80,70,60,50,40,30,20,101,0 CPr (mg/ml)1,02,03,04,05,06,07,08,09,01,00 TTGornal (ml) 44444444444 20 Lychnhxc0,3mlDCthm0,7mlmPBSpH7,4v4mlthuc thGornalvongnghim.Lcu,ccng30phtnhitphng, sau em o mt quang bc sng 550nm, cuvet 1cm. Tnh ton hm lng protein: Cc h s Ki l t l gia nng protein (mg/ml) v hp th quang: Ki =nng prute|nmt quang Tnh h s Ktb :Ktb = K|n|=1n (n: tng s mu chun, vi th nghim trn n = 10) Tnh nng protein theo mt quang:Cpr = Ktb D Pr = Cpr V Trong : D: hp th quang ca mu th V: th tch mi phn on protein Sau khi o quang ph hp th ca cc dung dch thu c kt qu sau. 21 Bng 2.2 Gi tr mt quang ca dung dch protein bit nng Cpr (mg/ml) 1,02,03,04,05,06,07,08,09,010,0 D0,060,1000,1470,1860,2250,2570,2900,3290,3620,410 K16,6720,0020,4121,5122,2223,3524,1424,3224,8624,39 Hnh 2.2 th tng quan gia nng protein v hp th quang. H s protein Ktb = 22,19 y = 0.0378x + 0.0287R = 0.997900.050.10.150.20.250.30.350.40.450 2 4 6 8 10 12Mt quangNng protein (mg/ml)OD 550ODLinear (OD)22 th thu c l ng thng c phng trnh y = 0,037x + 0,028 v c h s tng quan R = 0,998 ( 0,99). Nh vy, ti bc sng 550nm, trongkhong nng 1mg/ml 10mg/ml, nng protein v mt quang c mi quan h tuyn tnh. xc nh nng protein ca mt dung dch c th s dng h s Ktb trn vi iu kin nng protein nm trong khong gii hn thch hp. 2.2.4. Phng php xc nh hot RNase Nhtrnhbyphntngquan,cnhiuphngphpxcnh hot enzym, trong ti ny chng ti xc nh hot bng cch s dng c cht c hiu ca RNase l ARN. ARN +RNase oligonucleotide + RNase Nguyntc:ARNhpthccibcsng260nm.hpth (OD260) ph thuc vo nng ca ARN. Khi c mt enzym, RNase s thu phnARNthnhccoligonucleotiddomtquanghctngln.Da vo s thay i OD260 ta o c hot enzym. Mt n v (v) hot RNase l lng RNase xc tc phn ng thu phn ARN lm tng mt quang hc ca dch phn ng ln 0,01 v OD sau thi gian phn ng l mt pht trong nhng iu kin xc nh. S tng mt quang o trn my quang ph Spectro UV VIS Double Beam PC Scaning Spectrophotometer UVD 2960 v c tnh theo cng thc sau: A = OD1' (+E) -OD0'' (-E) . k -OD (E) Trong : + OD0'' (-E) l hp th ca (3000 -x) l dung dch c cht (ARN) 23 khi cha cho x l dch enzym vo. +OD1'(+E)lhpthcadchhnhpphn ngtnhtthi im sau 1' k t khi c x l dung dch enzym vo dung dch c cht. +OD(E)lhpthca(3000x)ldungdchmsaukhi thm x l dch enzym vo. + k l h s hiu chnh: ti thi im 0'', gi tr OD260 cho ch l hp th ca (3000 -x) l dung dch c cht, gi tr thc ca hn hp phn ng tnh ti thi im 0'' phi l hp th ca 3000 l dung dch c cht do vy k c xc nh nh sau: o OD260 ca (3000 -x) l dung dch c cht, k hiulOD0''S.Sauthmvoxldungdchmphaccht,gitro c k hiu l OD0''S+buf , khi : k = OD0'' S+buf / OD0"S Hot ring ca enzym (HRring) c xc nh bng: s n v hot caenzym/mgprotein.HmlngProteincxcnhbngphng php Biuret. Khi hot enzym RNase c tnh theo cng thc: HR = A,1 - Tin hnh:Pha dung dch c cht: cn 0,02 mg c cht pha trong 50 ml dung dch mPBSpH7,4haydungdchH2SO40,25N.Phalongdungdchtrn25 ln bng dung dch m tng ng. Lychnhxc0,1mlDCthm3mldungdchcchtARN.Lcu, o quang sau mt pht phn ng k t khi enzym tip xc vi c cht. 24 CHNGIII. THC NGHIM, KT QU V BN LUN 3.1.KTQUCHITTCHRNASETTRNGCHVTRNG CC3.1.1. Chit RNase t trng ch - Mc ch: Chuyn enzym t trong t bo trng ch vo cc dung dch m PBS pH 7,4 v dung dch H2SO4 0,25N. - Kt qu: Thu c 74 ml DCmTEa v 76 ml DCmTEb t trng ch. -Nhnxt:Dchchitthuctmitrng acidH2SO40,25Ntrong hn dch chit thu c t mi trng PBS pH 7,4.3.1.2.Nhnccphnonproteintdchchitmtrngchbngk thut ta phn on vi (NH4)2SO4 - Mc ch: Tm ra vng nng mui ti u ta protein - enzym t trng ch. -Ktqu:nhncttctmDCTE,trongcbnDCTEav bn DCTEb. Trong cc phn on protein enzym thu c u tin hnh o hm lng protein v hot enzym. 3.1.3. Chit RNase t trng cc - Mc ch: Chuyn enzym t trong t bo trng cc vo cc dung dch m PBS pH 7,4 v dung dch H2SO4 0,25N. -Ktqu:Thuc55mlDCmTCa v56mlDCmTCbttrngcc. Trongccphnonproteinenzymthucutinhnhohmlng protein v hot enzym. - Nhn xt: Dch chit RNase t t bo trng cc thu c mi trngacid H2SO4 0,25N trong hn dch chit thu c mi trng PBS. 25 3.1.4.Nhnccphnonproteintdchchitmtrngccbngk thut ta phn on vi (NH4)2SO4 - Mc ch: Tm ra vng nng mui ti u ta protein - enzym t trng cc. - Kt qu: nhn c tt c tm DCTC, trong c bn DCTCa v bn DCTCb.Trongccphnonproteinenzymthucutinhnhohm lng protein v hot tnh enzym. 3.2.KHOSTHMLNGPROTEINTRONGTRNGCHV TRNG CC nhlngproteintrongccphnondchchitbngphng php Biuret (k thut Gornal). - Mc ch: Xc nh phn on dch chit cho hm lng protein cao nht v s dng tnh ton hot ring ca enzym. Hm lng protein t trng ch Bng 3.1 Hm lng protein t cc phn on dch chit trng ch P PrTE-1aTE-2aTE-3aTE-4aTE-1bTE-2bTE-3bTE-4b D 0,1550,1830,1970,2050,0950,1540,2080,230 Pr (mg) 17,2020,0321,8522,7510,5517,1023,1025,50 26 -Nhnxt:HmlngproteinthuctccphnonDCtrongchai mitrng acidH2SO40,25NvmitrngPBSpH7,4khunhau,ch c phn on TE-1a thp hn ng k so vi cc phn on khc. Phn on (NH4)2SO4 bo ha 80% cho hm lng protein cao nht. Hm lng protein t trng cc Bng 3.2 Hm lng protein t cc phn on dch chit trng cc P PrTC-1aTC-2aTC-3aTC-4aTC-1bTC-2bTC-3bTC-4b D0,1670,2400,2760,3030,2350,3020,3110,361 Pr (mg) 18,5526,6530,6033,6026,0533,5034,5040,70 -Nhnxt:Hmlngproteinthuctngdntheomc(NH4)2SO4 bo ha. Trong , hm lng protein ca cc phn on dch chit trong mi trng acid H2SO4 0,25N thp hn trong mi trng PBS pH 7,4. 3.3.KTQUXCNHHOTRNASETTRNGCHV TRNG CC 3.3.1. Kt qu xc nh s phn b hot RNase t trng ch - Mc ch: RNase ng vt lng c mi ch c tm thy mt s 27 loi. Do , vic xc nh hot ca enzym cho php s b khng nh s cmtcaRNasetrongtbotrngch(Ranarogulosa.,Ranidae)Vit Nam. Kt qu xc nh hot RNase trong 4 phn on protein thu c t hai loi DCTE c tm tt trong cc bng sau. Bng 3.3Kt qu xc nh hot RNase trong cc phn on dch chit protein nhn c t DCTEa P PrV, mlPr, mgHR HR% theo cc ph HRring, U/mg TE-1a 517,20459,632,62 TE-2a520,035211,132,60 TE-3a521,8514531,056,64 TE-4a522,7522548,189,90 - Nhn xt:HR v HRring tng dn t phn on TE-1a n TE- 4a. PhnonTE-1avTE-2acHRvHRring gnbngnhau.Phnon TE-3a v phn on TE- 4a cho HR v HRring cao hn nhiu so vi 2 phn on TE-1a v TE-2a. Phn on TE-4a cho HR v HRring cao nht. HRring 28 tng dn theo mc (NH4)2SO4 bo ha. Bng 3.4 Kt qu xc nh hot RNase trong cc phn on dch chit protein nhn c t DCTEb P PrV, mlPr, mgHR HR % theo cc ph HRring, U/mg TE-1b 510,5519516,3918,48 TE-2b517,1021017,6512,28 TE-3b523,1028523,9512,34 TE-4b525,5050042,0119,60 - Nhn xt: HR ba phn on u gn bng nhau, phn on TE-4b

cho HR v HRring cao nht. Cc phn on u cho HRring kh cao. Phn on TE-2b v TE-3b c HRring gn bng nhau. So snh kt qu chit tch v phn b hot enzym RNase ca trng ch trong hai mi trng dd H2SO4 0,25 N v mi trng PBS pH 7,4 ta thy hotvhotringenzymtrongmitrngbazecaohntrongmi trng acid. c bit hot ring trong mi trng base cao hn nhiu mi trng acid. Cc phn on dch chit trong hai mi trng dd H2SO4 0,25 N 29 vmitrngPBSuchotvhotringtngdntheostng nng (NH4)2SO4 bo ha. 3.3.2.Kt qu xc nh s phn b hot tnh RNase t trng cc -Mcch:XcnhRNasectrongtbotrngcc(Bufosp., Bufonidae). Kt qu xc nh hot RNase trong 4 phn onprotein thu c t hai loi DCTC c tm tt trong cc bng sau. Bng 3.5Ktquxcnhhot RNasetrong ccphnondchchit protein nhn c t DCTCa PhV, mlPr, mgHR HR,% theo cc ph HRring, U/mg TC-1a 518,8573025,3928,75 TC-2a 526,6576026,4328,76 TC-3a 530,60110538,4336,11 TC-4a 533,602809,758,33 - Nhn xt: HR v HRring ba phn on u gn nh nhau trong phn on TC -3a cao nht. HR v HRring ca TC-4a l thp nht, thp hn nhiusovibaphnontrctuyhmlngproteincaTC-4acaonht 30 trong bn phn on. Bng 3.6 Kt qu xc nh hot RNase trong cc phn on dch chit protein nhn c t DCTCb PhV, mlPr, mgHR HR,% theo cc ph HRring, U/mg TC-1b 526,05126023,8648,37 TC-2b 533,50131524,9139,25 TC-3b 534,50251547,6372,90 TC-4b 540.701903,64,67 Nhnxt:HR tronghaiphnonuTC-1bvTC-2bgnnh nhau, phn on TC -3b c HR v HRring cao nht. Phn on 4 c HR v HRring thp nht. So snh kt qu chit tch v phn b hot HR t trng cc trong hai mitrngtathyHRvHRringtrongmitrngbazecaohnmi trng acid. Cc phn on dch chit trong dd H2SO4 0,25 N v dd PBS pH 7,4 ca trng cc u cho kt qu hot v hot ring tng dn t phn on(NH4)2SO4boha20%n60%vphnon(NH4)2SO4boha 60% l cao nht, n phn on (NH4)2SO4 bo ha 80% li thp nht. 31 BN LUN1. V chit tch RNase t trng ch v trng cc Trng ng vai tr quan trng trong sinh sn ca ng vt. Do , tng hp protein t bo trng din ra rt mnh m. Dn ti, t bo trng c hm lng protein thng cao hn cc loi t bo khc trong c th. iu s to iu kin thun li cho chit protein enzym. Tuy nhin, t bo trng c nhiu loienzymthamgiavoqutrnhtnghpproteinduytrbgencaloi, gykhkhnchotinhschproteinenzym.Ngoira,trnglpngvt lng c c v nhy bao bc bn ngoi bo v trng tt hn nhng cng gynhiutphntrongchittchenzym.Trongtrngccloingvt lng c cn c cc tp khc nh: glucid, lipid mng. Cc nghin cu v trng ch v trng cc Vit Nam cn cha nhiu. Saukhitmhiutiliuvccphngphpmitrongiutrungth, chngtitmthyhottnhgyctbocaRNaseckhnngp dngtrongiutrungth.Bivy,chngtimnhdnsdngcc phng php chit tch enzym b mn Ha Sinh, trng i hc Dc H Nitinhnhkhosthmlng,hotRNasettrnghailoing vt lng c l ch v cc Vit Nam.Mttrongccphngphptchenzymtruynthng,bovc enzyml sdngmui trung tnh (NH4)2SO4cc nngbo hakhc nhaukttaproteinenzym.Phngphpnytnhhngncutrc enzym. c bit, (NH4)2SO4 c mc ha tan trong nc cao (720g/l 25o C),tlmmthottnhenzym,trongnhiutrnghpcngipnnh enzym m gi thnh li thp nn c s dng nhiu trong thc nghim. Tuy nhin,phng phpnycmtsnhcimnh:mtnhiuthigian,ly 32 tm thu ta protein cn tc cao v nhit thp. Mt khc, mc tinh sch ca phng php thp v cn tin hnh loi mui ra khi ta enzym bng phng php thm tch hoc sc k lc gel. Thc t, trong gii hn cho php v thi gian v vi mc ch kho st banu,chngtitinhnhchittchRNasebngkttaphnon enzym protein vi mui (NH4)2SO4 cc nng bo ha khc nhau. Tuy k thut chit tch n gin, d lm, chi ph thp nhng chng ti cng thu c 16 phn on ta enzym protein c hm lng khc nhau bng 3.1 v bng 3.2. 2.V xc nh hot RNase ca hai loi trng ch v trng cc Nhtrnhbyphntngquan,cnhiuphngphpxcnh hot enzym. Theo mt s nghin cu tin hnh [7], [8], trong ti ny chng ti xc nh hot RNase bng cch s dng c cht c hiu l ARN nm men mua ca hng Sigma c cht lng cao. Enzym c bn cht l protein nn d dng b bin tnh, cn tin hnh o hotngaysaukhichittchvtrnhccyutcthgybintnh proteinenzym.CchtARNdbthyphn,cththyphnngaytrc khi tip xc vi enzym. Do , cn phi ch bo qun ARN trong ti nhm nhitthp,chphacchtngaytrckhiohotvthaotctin hnh nhanh chng. hn ch kh nng RNase b c ch hot ng, trnh s dng cc dng c th nghim c ion kim loi nng.Saukhiohotca16phnondchchitenzymthuthu c kt qu chng minh s c mt ca RNase. Hot ring ca RNase t trngccchoktqucaohnHdRringttrngch.Hotringcaonht ca RNase t trng ch l 19,60 U/mg nng (NH4)2SO4 bo ha 80% v t 33 trngccl72,90U/mgnng(NH4)2SO4boha60%.cbitls gimmnhHdRringttrngccnng(NH4)2SO4boha80%sovi cc nng bo ha khc. Trong hai mi trng chit, mi trng m PBS pH 7,4 cho HdRring cao hn mi trng acid H2SO4 0,25 N. Do , nn tin hnh chit tch RNase t trng ch v trng cc trongmi trng m PBS pH7,4.NntchRNasebngmui(NH4)2SO4 c nng bo ha cao,bo ha (NH4)2SO4 80% trng ch v 60% trng cc. 3. V ngha ca nghin cu Ungthlmttrongnhngcnbnhiutrphc tpnht.Ungth c th iu tr bng cc phng php nh: phu thut, x tr liu, ha tr liu, min dch tr liu hay cc phng php khc. Bi v, t bo ung th l t bo ca c th con ngi nn nhiu phng php iu tr tiu dit t bo ung th c nguy c ph hy c nhng t bo khe mnh trong c th. Phng php iu tr nhm ch s dng cc cht c kh nng tn cng mt cch c hiu i vi cc t bo ung th, gim thiu vic gy tn thng cho nhng t bo bnh thng ca c th. Do , gim bt c tc dng ph khi iu tr. Kh nng phn bit nhng t bo ung th v t bo bnh thng m ra hy vng mi trong iu tr cho bnh nhn ung th c bit l ung th no.Hot tnh gy c t bo chn lc trn t bo ung th l mt c im rt qu ca RNase. Amphinase l mt loi RNase loi ch (Rana pipiense., Ranidae)ckhnngtmkim,xmnhpvotboungth.Amphinasenhn ra lp ng c th trn b mt t bo ung th v bm cht vo , sau xmnhpvobntrong,thyphnARNgyctbo.Hottnhgy c t bo chn lc v kh nng xm nhp vo t bo ung th cc v tr su mrahng imi trong iu tr ung th nomtloiung th c bit 34 n l kh iu tr. Trong khi , cc cn b Dc hc cn t ch ti enzym ny. Vit Nam, ch v cc l ngun nguyn liu phong ph, di do. Bi vy, chng ti tin hnh nghin cu ngun Ribonuclease hai loi ng vt lng c l ch vccncta.Nghincuchmangtnhkhostsbbanungun Ribonucleasev cng thu c nhng kt qu nht nh. T , gp phn a racc hngnghin cu ngdng RNase iutr ung th nc ta trong tng lai. 35 KT LUN V XUT Kt lun Qua qu trnh nghin cu, ti thu c mt s kt qu sau:- chit tch c 16 phn on enzymth t t bo trng ch v trng cc bng (NH4)2SO4 c nng bo ho ln lt l 20%, 40%, 60%, 80% hai mi trng PBS pH 7,4 v mi trng acid H2SO4 0,25 N. - Cc phn on enzym th thu c trong c hai loi t bo trng ch v trng cc u c hot ca enzym RNase khc nhau. -thucccphnonenzymthttbotrngch,phnondch chit tch bng (NH4)2SO4 bo ha 80% mi trng m PBS pH 7,4 t hot ring cao nht l 19,60 U/mg. -thucccphnonenzymthttbotrngcc,phnondch chit tch bng (NH4)2SO4 bo ha 60% mi trng m PBS pH 7,4 t hot ring cao nht l 72,90 U/mg. xut - Tip tc nghin cu, kho st ngun enzym RNase t trng ch v trng cc Vit Nam. - Nghin cu gi tr s dng, ng dng enzym RNase t trng ch v trng cc, tintithnghimhottnhsinhhccaRNasesmpdngvoyhc. TI LIU THAM KHO Ting Vit 1.La K Hong, Trng Anh (1998), Cm nang bi thuc hay cho cc bnh thng gp, Nxb H Ni, H Ni, tr. 845. 2.NguynHuChn(1983),EnzymevxctcSinhhc,NxbYhc, H Ni. 3.PhmThTrnChu,NgcLin(1972),EnzymeI,II,ihc Tng hp, H Ni.4.Nguyn c Lng (2004), Cng ngh enzym, Nxb i hc quc gia thnh ph H Ch Minh, tp H Ch Minh, tr. 67 69.5.Nguyn Vn R (2002), Nghin cu to ch phm protease ngun gc ng vt, thc vt, ng dng trong phng chng suy dinh dng, Lun nTin sDchc, TrngihcDcHNi,HNi,tr.34 37, 43 46, 53 54.6.NguynXunThngvcngs(2009),Hasinhhc,NxbYhc, H Ni, tr.135 137, 169 172, 190. 7.NguynVnThit(2002),Nghincuhottnhribonucleolyticca nc rn h mang, Tp ch dc liu,7(6), tr. 181 185. 8.ng Th Thu, L Ngc T (2012), Cng ngh enzym, Nxb Khoa hc v k thut, H Ni, tr. 25 56.9.NguynVnThit,NgThHiYn(2003),Mtstnhchtc trng ca Ribonuclease t nc rn h mang en Vit Nam (Naja naja), NhngvnnghincucbntrongKhoahcssng.Boco khoa hc Hi ngh ton quc ln th hai, Nxb Khoa hc v k thut, H Ni, tr. 515 519. Ting Anh 10. A. Illanes (2008), Enzyme Biocatalysis Principles and Applications, Springer Science Publishing, p. 58 59. 11. Alfacell(2007),Ranpirnase:AmphibianRibonucleaseA,P-30 Protein Alfacell, Drugs in R & D, 8(2), tr. 120 124. 12. ArdeltW.,ArdeltB.DarzynkiewiczZ.(2009),Ribonucleasesas potentialmodalitiesinanticancertherapy,EurJPharmacol,625,tr. 181189.13. Ardelt W., Mikulski S. M., Shogen K. (1991) Amino acid sequence of an anti-tumor protein from Rana pipiens oocytes and early embryos. Homology to pancreatic ribonucleases, J Biol Chem, 266, tr. 245251.14. ArdeltW.,ShogenK.,DarzynkiewiczZ.(2008),Onconaseand Amphinase, the Antitumor Ribonucleases from Rana pipiens Oocytes, Curr Pharm Biotechnol, 9(3), tr. 215 225. 15. BabkinaG.T.,VasilenkoS.K.(1964),Nucleaseactivityofthe venoms of Mid- Asia Snakes, Biokhimiya, 29(2), pp. 268 272.16. BlzuezM.,FominayaJ.M.,HofsteengeJ.(1996),Oxidationof sulfhydrylgroupsofribonucleaseinhibitorinepithelialcellsis sufficient for its intracellular degradation, J Biol Chem, 271, tr. 18638 18642.17. CuchilloC.M.,NogusM.V.,RainesR.T.,(2011). "Bovine pancreaticribonuclease:Fiftyyearsofthefirstenzymaticreaction mechanism", Biochemistry, 50 (37), tr. 7835 7841. 18. DixonM.,WebbE.C.(1979),Enzymes,3rdediton,LongmanGroup Ltd., New York. 19. Gui-ZhiCheng,Jian-YuanLi,[...],andGuang-XiaShi(2009), Humanribonuclease9,amemberofribonucleaseAsuperfamily, specifically expressed in epididymis, is a novel sperm-binding protein, Asian J Androl, 11(2), tr. 240 251. 20. HaigisM.C.,KurtenE.L.,RainesR.T.(2003),Ribonuclease inhibitorasanintracellularsentry,NucleicAcidsResearch,31,pp. 31024 31032. 21. Hamann K. J., Barker R. L., Loegering D. A., Gleich G. J. (1987), Comparativetoxicityofpurifiedhumaneosinophilgranuleproteins for newborn larvae of Trichinella spiralis, J Parasitol, 73, tr. 523529. 22.HarderJ.,SchroderJ.M.(2002),RNase7,anovelinnateimmune defenseantimicrobialproteinofhealthyhumanskin,JBC,277(48),pp. 46779 467784.23. HuangH.C.,WangS.C.,LeuY.J.,LuS.C.,LiaoY.D.(1998), TheRanacatesbeinarcrgeneencodingacytotoxicribonuclease: Tissuedistribution,cloning,purification,cytotoxicityandactive residues for RNase activity, JBC, 273, pp. 6395 6401. 24. KimJ.S.,SoucekJ.,MatousekJ.,RainesR.T.(1995),Catalytic activityofbovineseminalribonucleaseisessentialforits immunosuppressive and other biological activities, Biochem J, 308(2), tr. 547550. 25. KobeB.,DeisenhoferJ.(1993),Crystalstructureofporcine ribonucleaseinhibitor,aproteinwithleucine-richrepeats,Nature, 366, tr. 751756.26. LelandP.A.,RainesR.T.(2001),Cancerchemotherapy-Ribonucleases to the rescue, JBC, 8, pp. 405 413. 27. Leland P. A., Schultz W., Kim B. M., Raines R. T. (1998), RNase Avariantswithpotentcytotoxicactivity,PNAS,95(18),pp.10407 10412. 28. LiuD., CardilloT.M., WangY., RossiE.A., GoldenbergD. M., ChangC.H.(2014),Trop-2-targetingtetrakis-ranpirnase has potentantitumoractivityagainsttriple-negativebreastcancer,Mol Cancer, 13, tr. 53. 29. MajchrzakA., WitkowskaM., MdraA., ZwoliskaM., Bogusz J., Cebula-ObrzutB., DarzynkiewiczZ., RobakT., SmolewskiP. (2013),Invitrocytotoxicityof ranpirnase (onconase)incombination withcomponentsofR-CHOPregimenagainstdiffuselargeBcell lymphoma (DLBCL) cell line, Postepy Hig Med Dosw, 67, tr. 1166 1172. 30. MikulskiS.M.,ChunH.G.,MittelmanA.,PanellaT.,PuccioC. A.,ShogenK.,ConstanziJ.J.(1995),Relationshipbetween response rate and median survival in patients with advanced non-small celllungcancer:comparisonofONCONASE withotheranticancer agents, Int J Oncol, 6, tr. 889 897. 31. MikulskiS.M.,ConstanziJ.J.,VogelzangN.J.,McCachrenS., Taub R. N., Chun H., Mittelman A., Panella T., Puccio C., Fine R., ShogenK.(2002),PhaseII trialofasingleweeklyintravenousdose of ranpirnase in patients with unresectable malignant mesothelioma, J Clin Oncol, tr. 27481.32. MikulskiS.M.,GrossmanA.M.,CarterP.W.,ShogenK., Costanzi J. J. (1993), Phase I human clinical trial of ONCONASE (P-30 Protein) administered intravenously on a weekly schedule in cancer patients with solid tumors, Int J Oncol, 3, tr. 5764. 33. Mosimann S. C., Ardelt W., James M. N. G. (1994), Refined 1.7 X-raycrystallographicstructureofP-30protein,anamphibian ribonuclease with anti-tumor activity, J Mol Biol, 236, tr. 11411153. 34. MuellerC.A.,SeymourR.S.(2011),Theimportanceof perivitellinefluidconvectiontooxygenuptakeofPseudophryne bibronii eggs, Physiol Biochem Zool, 84(3), tr 299 305. 35. PavlakisN.,VogelzangN.J.(2006),Ranpirnaseanantitumour ribonuclease:itspotentialroleinmalignantmesothelioma,Expert Opin Biol Ther, 6, tr. 19. 36. Pelmont J. (1993), Enzyme, Presses universitaires de grenobe. 37.RainesR.T.(1999),RibonucleaseA:frommodelsystemtocancer chemotherapeutic,Enzymaticmechanism,IOSpress,Washington,DC,pp. 235 249 . 38. Rosenberg H. F., Dyer K. D. (1995), Human ribonuclease 4 (RNase 4):codingsequence,chromosomallocalizationandidentificationof twodistincttranscriptsinhumansomatictissues,NucleicAcidsRes, 23(21), tr. 4290 4295.39. SandeepKGupta,BrendanJHaigh,FrankJGriffin,ThomasT Wheeler(2013),ThemammaliansecretedRNases:Mechanismsof action in host defence, Innate Immun,19(1), tr. 86 97. 40. SinghU.P., ArdeltW., SaxenaS.K., HollowayD.E., Vidunas E., LeeH.S., SaxenaA., ShogenK., AcharyaK.R.(2007), Enzymaticandstructuralcharacterisationof amphinase,anovel cytotoxic ribonuclease from Rana pipiens oocytes, J Mol Biol, 371(1), tr. 93 111. 41. SmolewskiP.,WitkowskaM.,ZwolinskaM.,CebulaObrzutB., MajchrzakA.,JeskeA.,DarzynkiewiczZ.,ArdeltW.,ArdeltB., RobakT.(2014),Cytotoxicactivityoftheamphibian ribonucleases onconase andr-amphinaseontumorcellsfromBcell lymphoproliferative disorders, Int J Oncol, 103892, tr. 2014 2405. 42. VasilenkoS.K.,BabkinaG.T.(1965),Isolationandpropertiesof the cobra venom nuclease, Biokhimiya, 30(4), pp. 705 712.43. Vlassov A. V. (1998), Human Ribonucleases, Biochemistry, 63(12), pp. 1349 1360. 44. WuY.N.,MikulskiS.M.,ArdeltW.,RybarkS.M.,YouleR.J. (1993), A cytotoxic ribonuclease: study of the mechanism of onconase cytotoxicity, JBC, 268(14), pp. 10686 10693. 45. YangD.,ChenQ.,RosenbergH.F.,RybakS.M.,NewtonD.L., Wang Z. Y., Fu Q., Tchernev V. T., Wang M., Schweitzer B., et al (2004),HumanribonucleaseAsuperfamilymembers,eosinophil-derivedneurotoxinandpancreaticribonuclease,inducedendriticcell maturation and activation, J Immunol, 173, tr. 61346142.46. Youle R. J., Wu Y. N., Mikulski S. M., Shogen K., Hamilton R. S., NewtonD.,DAlessioG.,GravellM.(1994),RNaseinhibitionof human immunodeficiency virus infection of H9 cells, Proc Natl Acad Sci USA, 91, tr. 60126016. PH LC 1. Dung dch m PBS pH 7,4 - Cn 8 g NaCl; 0,2g KCl; 3,58g Na2HPO4; 0,24g KH2PO4 pha trong 1l nc ct. - o kim tra li pH ca dung dch. 2.Phadungdchcasein1%:hatan1gcaseinhydrocloridtrongdungdch m PBS pH 7,4. 3. Pha thuc th Gornal - Ha tan 1,5g CuSO4.5H2O v 6,0g Na K tatrat trong 500,0ml nc ct. - Thm 300,0 ml dung dch NaOH 10%, va thm va lc u. - Thm 1g KI ri b sung nc ct va 1000ml. - Lc k, bo qun trong chai thy tinh c mu. 4.DungdchacidH2SO40,25N:rtcnthn7,5mlacidH2SO4 cvo 100ml nc, lc u, ngui n nhit phng, pha long vi nc va 1000ml