• Nghien
CIFU- Ky thuat
khae chira boat c h i t perindopril tert-butylamin va indapamid.
Tai lieu tham khao
1. European Pharmacopoeia 8.0, pp. 2480-2482, 2991-2994.
2. Janardhanan Anand Subramony (2010), United States Patent application publication, Perindopril fomulations, US/2010/0076052 A l , pp. 1-14.
3. Impurities of Indapamide, Bioindustria L.i.M S.p.A (2010), pp. 1-2.
4. Jogia H, Khandelwal tJ, Gandhi T, Singh S, Modi D. (2010), "Development and validation of a stability- indicating assay method for simultaneous determination of perindopril and indapamide in combined dosage forni by reversed-phase high-performance liquid chromatography", J.AOACInt, 93(1), 108-15.
5. Hari Hara Theja Dugga, Ramalingam Peraman, Devanna Nayakanti (2013), "Stability-Indicating RP- HPLC method for the quantitative analysis of perindopril
{Ngay nhan bai: 18/11/2015
erbumine in tablet dosage form", J. of Chromatographic Science, 2013, 1-6.
6. Lakshmana Rao, S.C. Dinda (2013), "Validated stability indicating reverse phase HPLC method for the simultaneous estimation of Perindopril and Indapamide in pharmaceutical dosage forms". International J. of Pharmacy 3(1), 277-289.
7.NevinErk(2001),"Comparisonofspectrophotometric and an LC method for the determination perindopril and indapamide in phannaceutical fonnulations", J. of Pharmaceutical and Biomedical Analysis, 26(1), 43-52.
8. Pathak, A. K., Lodhi, Rachana, Shikhri, Mukesh K. (2011), "A selective reverse phase HPLC method for simultaneous determination of perindopril and indapamide in solid dosage form and bulk drug", J. of Pharmacy Research, 4(8), 2512.
9. ICH-Q2(R1) (2005), Validation of analytical procedures: Text and methodology, pp. 1-13.
10. Bp Y te (2015), Thdng tu quy dinh viec dang ky thuoc: Huang din cua Asean ve tham dinh quy trinh phan tich, tr.26-\-274.
•Ngay duyet dang: 25/04/2016) /
Nghien cmi phan tach, xac dmh hoat do I va mot so tinh chat cua ribonuclease trirng ecn
(Rana rugulosa Wiegmann)
Nguyen Quoc Toan', Nguyen Van Rir^*
' Vu Bao hiem y te - Bg Y te
-Truang Dai hoc Duac Hd Noi 'E-mail: [email protected] SummaryRibonuclease (RNase) of the eggs of the frog (Rana rugolusa), known having cytotoxicity, therewith possessing strong antitumor activity, anti-inflammatory and antivims, was isolated. The isolation was optimized to gain high yield with BPS buffer, incubated at37°C for 30 minutes. The obtained extracts showed the desired specific activity of 3.27 ±0.12 U/pg protein, being significantly higher in BPS buffer condition than in acid medium (2.72 ±0.16 U/pg protein) (p < 0.05). Purification and identification of some enzymic properties of the extract B was performed with ammonium sulfate (30-40% saturated), G25 sephadex, by ion exchange chromatography, polyaerylamide electrophoresis in combination of pressed agarose gel. Thereby, the RNase of the frog eggs was obtained in high purity and of specific activity 40 - 60 U/ml, with 12 kDa in molecular weight These all were comparable to those of the reference.
Keywords: Ribonuclease (RNase), activity, properties, cytotoxicity, frog eggs.
Dat van de
Trirng eiia I c h ddng cd ten khea hpc la {Rana rugulosa Wiegmann) da d u g c b i l t la cd heat tinh gay dpe t l bae "'. Trirng I c h cd ribonuclease (RNase) dang d u g c nhieu nha khoa hpc tren t h i gidi quan tam nghien cu-u ["l Ngeai kha nang diet t l bao ung t h u , RNase edn ed nhu-ng tac dung
sinh hpe quan trpng khae n h u khang khuan, khang virus,...I''-1. a Viet Nam, ngudn d u p h i m tru-ng trcng e h l bien I c h lam thge p h i m r i t phong phu, ed t h i la nguyen lieu ddi dao cho nghien ciru c h i l t tach RNase. D l gdp p h i n khai thae ngudn d u pham lam thudc chu-a benh i=i, muc tieu eua nghien cipu la: c h i l t tach, xac djnh hoat dp va met sd tinh c h i t eiia RNase phan lap tip trirng I c h .
TAP CHI DtTOC HOC - 5/2016 (SO 481 NAM 56) 51
Nghien CLPU - Ky thuat
Nguyen lieu va phu'O'ng phap nghien CLPU Nguyen lieu
£ch ddng {Rana rugulosa Wiegmann), hp £ch (Ranidae) thu gem d khu vgc Ha Ndi, tach liy tn>ng, bac quan d nhiet dp -20°C trong tii lanh am sau.
Hoa chat
ARN tuyp VI, Sigma; nude tinh khiit khdng cd RNase va DNase (H^O deion); dung djeh (Dd) RNase chuin (S) 10 mU/ml; thudc thir Bradford;
Ceomassie brilliant blue G250; aerylamid; TEMED;
marker (M) unstained protein MW 12 kDa; marker pageruler prestained protein 15 kDa va marker miu 70 kDa, Hang Fermentas; dung djch dem running buffer; dung djeh rira mau; agarose 0,8%;
dem TAE; ethidium bromid.
Dung cu va t h i l t bj
Pipetman, Ing Falcon, Eppendorf, can phan tieh 4 sd, may nghiin ddng t h i , ddng khd 1-2 LD PLUS, may do pH 210, edt Sephadex G25 (Biotech), may do quang pho IMPLEN nanephetometer P330, may PCR PTC-100 Peltier Thermal Cycler, may ly tam lanh Sen/all Legend RT, may sac ky FPLC AKTA, sac ky trao doi cation Hitrap SP XL 1 ml, may dien di dung Mini Trans- Blot cell, dien di ARN Mupid-2 Plus Advance, may sei ARN - UV - Transilluminator.
Phu'O'ng phap nghien cii-u Xac djnh hoaf do RNase
ARN hip thu cue dai d A 260 nm, dd hip thu (ODjgj,) phu thude vao ndng dp phan tir ARN.
Trpng dung djch ARN bj RNase thuy phan thanh cac pelynucleotid nhd hon gay tang OD. Do sg thay ddi eiia OD^g^. Don vj heat dp RNase (U) la lugng RNase xue tac thuy phan ARN tdng s l trong dieu kien xae djnh lam tang OD^^^ dung dich phan ung len 0,01 don vj sau phan irng 60 giay
Hoat do RNase (A) tinh theo edng thire:
Miu trang (W) la hdn hgp phan ung enzym vdi CO chit (E-i-S) tai thdi dilm 0 giay.
Hoat do rieng (HDR) eiia RNase (A J: la s l don vj hoat dp RNase (U) cd treng 1 pg protein c h i phim; Ham lu-gng pretein xac djnh bing phugng phap Bradford i2-''i.
Ky thuat dijc Id tren gel agarose 0,8% xac dinh hoat do RNase
Diing ong falcon liy 10 ml gel agarose 0,8%
hda tan trong 100 ml dem TAE IX, dun sdi ndng chay them 50 pi Dd ARN nong dp 10 mg/ml, trdn deu va dd khudn, de gel ddng lai. Diing dng hut da khir trung due Id tren ban gel. Bom 10 pl/miu
thir vao cae Id da due theo quy hoach va thir tg danh dau (tu-1 -10); Chu-ng (-) hoae trang (w): la mdi trudng dem hoae hon hgp E + S chua phan irng) tuong u-ng vdi timg loai thir; M i u chu-ng {•*•) hoae ehuan (S): Dd RNase A ndng dp 0,1 mg/ml. 0 ban gel d 37°C, 30 phut, nhudm ethidium bromid 7 phiit, soi tip ngoai va dgc k i t qua tren may.
Chief tach finh che RNase: Dga theo ham s l f(xij) eua phuong trinh hoi quy:
n n n f ( x ) = b + I b x + I b . x . x + I b . x 2
^ i j ' o I I U I J 11 I
i=1 j=1 1=1
Ghi chii: b^ 6 b,^ b„: he sd hdi quy; x,x la yeu to tac dong;
i=1^n,j=1->n, i^j, nia so yeu td.
Cf eae thf nghiem treng nghien eu-u nay heat dp RNase dae hieu rieng (HDR A^) la ham sd f(x..) phu thude eae blln sd x trong dd: b, - lugng nguyen lieu la djeh ehilt trirng Ich la hang sd (In djnh mpt lugng nhit djnh); dllu kien chilt tach: x, - pH acid (A) heac pH kilm (B); x^- gia trj nhiet dp tae ddng;
Xj - thdi gian thir nghiem ( 5 , 10, ... 60 phiit); x^- tae nhan kit tiia (% amoni sulfat bao hda), x^- dllu kien sac ky. A„ (U/pg Pr) = f(x,) = max la gia trj HDR A^ eao nhit thu dugc tu- mdt qua trinh thi nghiem nhd Iga chpn hang sd (bij) va eae bien s l (xij). Ma thi nghiem eho k i t qua HDR A^ cao nhit treng tap hgp thi nghiem (n^ > 3). Khae sat qua trinh thge nghiem thu duge cac gia trj trung binh v l HDR A„ eiia RNase la X,^ = Xi ± SD P-^).
Nghiin dong the va ly tam tao djch chief trirng ech (DCTE) 1/5: Trimg leh (10 g) nghiin ddng t h i vdi 50 ml Dd dem, ly tam d 10.000 vdng/
phiit treng 20 phut d 4°C, loai can va thu dich ehilt DCTE^(vdi Dd H^SO^ 0,25 N) va DCTE^ (vdi dem PBS).
Ket tua phan doan protein enzym: Sir dung
% amoni sulfat bao hda (% ASBH) Sac ky cot: sir dung sephadex G25
Sac ky trao doi ion flnh che RNase: Sir dung he thong FPLC AKTA.
Thir tinh bin nhiet eiia RNase: SLP dung may PCR d l gia nhiet trong 5 phut. Xac djnh boat dp RNase, tim nhiet dd leal tap tdt nhit va thu dugc A max.
0
Xac dmh nhiet do hoat dgng toi uu (f^^J va pH hoat dgng foi uu (pH„^,): Dung may PCR d l il hon hgp miu. Do OU^^ cua miu d pH tip 1,5 - 9.
Dien di polyacrylamid khong bien tinh ket ha'p ep gel agarose: Pha gel polyacrylamid khdng ed SDS gdm: gel tach (16%) va gel cd (5%). Pha m i u thir tu cac k i t tiia bang Dd dem da sip dung trudc dd thanh mdi m i u 5 ml;
52 TAP CHI DlTOC HOC - 5/2016 (SO 481 NAM 56)
• Nghien ciru - Ky thuat
hilt 10 pi moi m i u thir vae 10 g i l n g thee thir t g 1 - 10 t u o n g u-ng v d i tua 10 - 80 % A S B H (so do k i t qua eae ban gel); chay d i n khi vach eiia Marker mau g i n cham mep d u d i ciing, dirng lai, thao ban gel. Ep trung kbit len tren gel agarose 0,8%, ii d 37°C trong 30 phut, tach ban gel polyacrylamid va nhudm gel bang ethidium bromid 7 phut, rira bang n u d c deien, dat ban gel nay len gel agarose, soi tir ngoai, xae djnh heat dd RNase.
Nhudm gel polyacrylamid bang ceomassie, 30 phut va rira b i n g Dd rira mau. Soi tren may, k i t hgp vdi gel agarose de xae dinh vj tri bang cd hoat tinh, kich t h u d c bang va boat dp RNase 121.
Xir ly so lieu: Sd lieu xir ly tren may tinh, p h i n m i m Excel 2013, test T-student. N g u d n g vdi dp tin eay 95%, s g khac biet ec y nghTa khi p < 0 , 0 5 .
K i t qua nghien CLPU Tao djch chiet DCTE ty le 1/5
Sir dung 10 g trirng l e h n g h i i n ddng the vdi 50 ml Dd H j S O , 0,25N (A) hoae 50 ml Dd dem PBS 0,1 M pH 7,4 (B), ly tam d 10.000 vdng/phiit treng 20 phut, bd can, hoan thien thee ty le trirng l e h / djch c h i l t la 1/5, thu d u g c DCTE^ va DCTE^
tuy theo dung dieh dem. Xae djnh heat dp RNase trong 2 dieh c h i l t b i n g ky thuat due Ic tren gel vdi RNA dae hieu. K i t qua d u g c trinh bay trong bang 1.
Phan tach RNase tip DCTE^ 1/5 bang phu-o-ng phap xir ly nhiet
T i i n hanh gia nhiet tren may PCR, 5 phut d cae nhiet dp, rdi ep gel agarose 0,8% d 37°C, 30 phut, nhudm m l u ethilium bromid 7 phut, k i t qua d u g c t h i hien tren hinh 1.
p
25'C
m
60=C
j f v
90'C
m-
DCTE^ . 40=C
70°C
•
100°C
m (-)
(S), 50=C
80=C
«
#
25=C
t^^^
'•^a^''-^
60^C
•
90°C
m
^^^^'^^'
wc
m
70°C
•
-ioo°c
•
50=C
• '
80°C
• . ( - ) ^ (S^Ok
Hinh 1 : Ban gel xae djnh hoat do RNase a trong djch ly tam tu DCTE
Chimg am (-) ciia OCTE^ sir dung Dd H^SO^ 0,25N va cua DCTE^ sir dung Dd PBS 7,4;
Chimg duang (•*•): sir dung Dd RNase A nong do 0,1 mg/ml.
Tip k i t qua tren hinh 1, xac djnh boat dp RNase, loai bd cac trj sd trung gian cd gia trj t h i p d tren
ban gel, se sanh tim k i t qua cd gia trj eao du-ge trinh bay tren bang 1
Mau thir 1a uj* 4a B 7a
8a 1b u j - 4b H 7b
8b Nhiet, °
25 60 iO 100
25 60 90 100 Banh gia
Bang 1:
C So TN, n 3 3 3 3 3 3 3 3
Hoaf dp RNase Prt6ng,pg 20,60 ±1,05 13,28 + 0,85 7,12 ±0,21 5,44 ±0,22 26,96 ±1,26 17,72 ±1,20 8,04 + 0,55 7,76 ± 0,35
trong djch ly tam HDRNase(A),U
243 ±1,25 23,7 ±1,28 19,4 +0,25
10,6 ±0,25 29,7 ±1,45 27,3 ±1,16 26,3 + 0,28 16,5±0,15
DCTE gia nhiet a 40-100'C/5 phiit
^i%' °/"
100 97,53 79,84 43,62 100 91,92 88,55 55,56%
HDRA^cf7ava7bdeuiJat
HDR (A J, U/pg Pr 1,18 ±0,25 1,78 ±0,20 2,72 + 0,16 1,95 ±0,31 1,10±0,18 1,54 + 0,17 3,27 + 0,12 2,13 + 0,10
"13>=-A„™>A™.P
HS: A^ tang, lin 1,00 1,51 2,31 1,65 1,00 1,40 2,97 1,93
<0,05
Y nghia A^,^
A - m a x A^ = max
A„,.
A - m a x A^ = max
TAP CHI DlTOfC HOC - 5/2016 (SO 481 NAM 56) 53
Nghien CCPU - Ky thuat
K i t qua bang 1 cho t h i y : DCTE^ xir ly nhiet d 90°C treng 5 phut da loai d u g c p h i n Idn cae pretein tap kem b i n nhiet va tach d u g e Dd RNase ed HDR A„ max dat 2,72 ± 0,16 U/pg Pr va he sd (HS) A^ tang 2,31 i l n so vdi khi chua xir ly nhiet (25°C),°DCTEg xir ly nhiet d i n 90°C eung loai d u g e cac protein tap va dat A^ max la 3,27 ± 0,12U/pg Pr, tang hon cd y nghTa se vdi DCTE^ vdi p < 0,05.
N h u vay DCTE^ ehilt d mdi trudng pH 7,4 dat hieu qua ve A^^ eae hgn d mdi trudng acid (pH 2).
Do dd, cac thi nghiem t i l p theo Iga chpn DCTE^ d l tinh e h l tae e h l p h i m enzym va xac djnh mdt s l tinh c h i t ciia RNase.
Xac djnh tho-i gian xu' ly thich hgp 6' 100°C de tach dirge toi iru hoat tinh RNase
T i i n hanh xac djnh boat tinh RNase trong cac m i u thir nhiet bang may PCR a IOOPC tip DCTEg tip 5 d i n 30 phut thu d u g c k i t qua trong hinh 2.
Hinh 2: Ban gel xac dinh hoat tinh RNase cdn lai trong djeh iy tam tir DCTE^
Chimg am (-): Dd PBS 7,4; Chimg duang (+); Dd RNase A nong do 0,1 mg/ml
Hinh 2 quan sat t h i y vdng hoat dp RNase xCr ly d 100°C/5 phut ed dien tieh khoanh trdn gan tu-ong du-ong vdi m i u chirng (+), k i t qua d u g c tnnh bay trong bang 2.
Bang 2: Hoat do RNase trong djch ly tam ciia DCTE. sau khi gia nhiet Stt
M
1 2 3 4 5 6
T,„„„«Phut DCTE,
5 10 15 20 25 30
Prt6ng,pg 10,42 ±1,26 10,28 +1,24 10,23 ±1,23 10,05 ±1,21 9,81 ± 0,87 9,43 ± 0,83 9,36 ± 0,78
HO RNase (A), U 23,1 ±1,34 23,1 ±1,45 12,6 ±0,91 10,2 ±0,83 9,4 ± 0,57 5,3 ±0,34 2,5 + 0,22
Ty/e.-A/4„j,%
100 89,53 48,84 39,53 36,43 20,54 9,69
HDR (A J, U/pg Pr 2,25 ±0,21 2,25 ±0,31 1,23±0,11 1,01 ±0,12 0,96 ± 0,09 0,56 ± 0,05 0,27 ±0,03
He so A^ tang, liin 1,00 1,94 1,06 0,88 0,83 0,48 0,23
Y nghia
>0,0S
<0,05
<0,05
<0,05
<0,05
<0,05 Bang 2 cho thay xir ly nhiet leai protein tap tu-
DCTEg la lOO-C, 5 phut la hgp ly.
K i t qua tren eho t h i y RNase tnj-ng I c h ehju dugc d 90 - 100°C trong khoang 5 - 1 0 phiit, chung td kha nang b i n nhiet cao, vi vay cd t h i Igi dung tinh c h i t nay d l nang nhiet dp len 90 -100°C gay k i t tiia, leal cac protein tap trong qua trinh tach ehilt,
Ket qua hoat do RNase trong tua phan doan Hinh 3: San gel xac djnh hoat tinh RNase trong theo % amoni sulfat bao hoa cac phan doan protein tie DCTE^
Xae djnh heat dp RNase trpng tiia phan dpan Chimg (-):Dd dem PBS 0,1 MpH 7,4.
protein tip DCTEg. Chimg (•^): Dd RNase 0,1 mg/ml Do mau va xae djnh heat dp RNase thu dugc k i t qua tren bang 3.
Bang 3: Ket qua xac djnh hoat do RNase trong cac phan doan protein tir DCTE^
Stt,i PdPr%ASBH(%) Prtong.pg HDRNase(A),U DCTE 7,12 ±0,25 19,4 + 1,45
Tyle:A,/Ai,,% HDR (A J, U/pg Pr HStangA,,lan S'nghia
10
100
2,72 ±0,13 1,000,37 ±0,C 2,1 ±0,21 10,82 20
5,68 ± 0,20 0,68 ±0,14 4,5 ± 0,42 23,20
30 6,62 ± 0,23 2,43
1,04 ±0,04 24,8 ±1,37 127,84 40
23,85 ±1,25 8,75
1,12*0,09 29,4 +1,46
50 0,98 ±0,13
151,55 26,25 ±1,36 ),63
11,5±1,05 60
59,28 0,84 ±0,11
70
5,3 ±0,23
11,73 ±0,96
431
27,320,83 ±0,15 3,2 ± 0,25
6.31 ± 0,27 2,32 16,49
0,46 ± 0,07 3,86 ±0,15 1,41
Dd sau £
2,5±0,15
0,32 ± 0,02 5,43 ±0,14 1,99
1,4 ±0,13 7,22 4,38 ±0,12 1,61
54 TAP CHI Dl/OC HOC - 5/2016 (SO 481 NAM 56)
Nghien CIFU - Ky thuat
K i t qua eho t h i y heat dp RNase chu y l u eua DCTEjj la k i t tua cua phan doan 30 - 40 % amoni sulfat bao hda (ASBH), dp vay chpn ndng dp nay de tach p h i n ldn RNase.
Sau 2 bude xu- ly nhiet d l leal protein tap va k i t tiia bang % ASBH che t h i y : protein thu d u g c da kha tinh saeh ed heat dp RNase dat gia tri cac. N h u vay, sau b u d c nay pretein eiia phan dean 30 - 4 0 % ASBH la nguon RNase d u g e dung d l t i i n hanh tinh e h l t i l p theo.
Xac djnh hoat do RNase trong cac phan doan tinh sach bang cot sephadex G25
Tiia phan dean theo % ampni sulfat bao hda d u g c hda tan lai bang 5 ml dem PBS pH 7,4 t u o n g u-ng vdi loai djeh d l c h i l t tach RNase. Xae dinh hoat dp RNase b i n g ky thuat due lc tren gel agarose, k i t qua t h i hien tren hinh 4.
Hinh 4: Kit qua hoat dd RNase trong cac phan doan qua edt sephadex 025
K i t qua cho t h i y m i u thip di qua cdt loai mudi sephadex G25 cd RNase chii y l u tap trung treng m i u thir F1 va ed r i t it d cae m i u thir khac (tu E1 din E5).
Phan lap va xac djnh hoat dp RNase bang sac ky trao doi ion
Phan doan protein (tach d u g e b i n g 30-40%
ASBH) tip DCTEjj sau khi da leai mudi d u g c s i c ky trao doi ion tren edt Hitrap SP XL che k i t qua tren hinh 5.
So phan doan
Hinh 5: Sie ky dd eua eae phan doan DCTE tren edt Hitrap SP XL
A: Hoat dd RNase (U) / CV (1 ml); Pr: Ham tuang protein (mAU)/CV (1 ml)
Duong cheo ia gradient ndng dp NaCI tif 0 din 1M.
Hinh 5 cho t h i y boat tinh RNase x u i t hien d eae phan doan tip 16 d i n 20 CV ( t h i tieh edt Hitrap SP XL = 1 ml). B i n g each gom cac phan doan tip 18 d i n 20 CV ed boat dp A^ eae (40 - 60 U/ml), tao d u g c c b l pham dung djeh RNase ed boat dp enzym tu- 40 - 60 U/ml va ed t h i t i i n hanh ddng khd tao e h l pham bdt.
Xac djnh mpt so tinh chat cua RNase trong DCTE
Xac djnh gia trj nhiet do foi iru (T^ ^ Sir dung may PCR ii hon hgp phan irng gdm c h i p h i m dung djeh RNase va ARN d nhiet dp t u 25 - 80°C trong 1 phiit, do ODj^j^ cae m i u thir va xac dinh hoat dp RNase (A), k i t qua thu d u g e t h i hien tren hinh 6.
A(U)
Hinh 6: Biin doi hoat do RNase cua dung djch RNase theo nhiet do
K i t qua ehe t h i y gia trj eae n h i t la 60 U/ml t u o n g irng vdi nhiet dd phan irng d 40 - 45°C, dd la nhiet dp hpat ddng tdi u u ciia enzym nay.
Xac dmh gia trj pH toi uu (pH^ ^
Tien hanh do OD^g^j eiia hon hgp phan u-ng RNase vdi ARN trong mdi tnj-dng dem natri phosphat 20 mM pH 6, nhiet dd 40 "C, sir dung Dd acid citric 0,1 M d l d i i u ehinh pH giam va Na^HPO^
0,1 M d l d l l u chlnh pH tang thee day gradient. K i t qua thu d u g e tren hinh 7.
A{U) SO 40 30 20 10
^ ^ H t f * - ^ ' ^ " ^ ^ *
pHopt
1 pH
0 1 2 3 5 6 7 8 9 10
Hinh 7: Biin ddi hoat do enzym cua dung djch RNase theo pH
K i t qua eho t h i y RNase trong DCTE^ ed boat tinh manh n h i t d pH tip 7,0 - 8,0.
TAP CHi DUOC HOC - 5/2016 (SO 481 NAM 56)
• Nghien cu'u - Ky thuat
Xac d j n h khoi lu-gng p h a n t u ' cua RNase T i i n hanh dien di eae phan doan (PD) tiia protein sau qua trinh phan lap b i n g amoni sulfat tu-1 - 8 (tuong ipng vdi tip 10 - 8 0 % bao hda AS) CLia DCTEg, Marker RNase (M) cd k h i i lu-gng phan tir la 12 KDa bang dien di polyacrylamid khdng b i i n tinh k i t hgp ep gel agarose. K i t qua xac djnh bang RNase tir DCTE^ d u g c b i l u thj tren hinh 8.
Hinh 8: Kit qua dien di xac djnh khdi lugng phan tir RNase thu duge tu DCTE^
K i t qua che t h i y bang RNase d cac phan dean 3, 4 t u o n g u-ng vdi khdi l u g n g la 12 kDa.
Cac bang RNase d phan dean 3 va 4 x u i t hien rd ret va h i u n h u khdng xuat hien v i t tap, chirng td qua trinh tinh c b l da tach d u g e RNase tinh saeh.
Ban luan
Sir dung mdi trudng dem PBS 0,1 M pH 7,4 cd pH d vung base g i n trung tinh, t u o n g t g vdi d l l u kien trong t l bao, d l tach c h i l t RNase tao d u g c djch chilt DCTEg dat 3,27 ± 0,12 U/ pg Pr, cae hon so vdi mdi trudng H^SO^ 0,25N chf dat 2,72
± 0,16 U/ pg Pr, s g khae nhau ed y nghTa thdng ke (p < 0,05). Vi vay chung tdi da chpn dem PBS nay trong sudt qua trinh ehilt tach va nghien ciru v l RNase.
Cac k i t qua treng nghien ciru nay da che t h i y RNase tach c h i l t tu- tru-ng l e h cd mdt sd tinh chat dae biet da d u g c xac djnh va sir dung de thuc hien treng ehinh qua trinh tach c h i l t va tinh c h i enzym n h u la tinh b i n nhiet eae, k i t tiia RNase ehii y l u tap trung d mdi trudng 30 - 40 % amoni sulfat bae hda t h i p , trong khi nhiet dd heat ddng tdi uu eua enzym nay t h i p (40 - 45°C), pH tdi uu nam treng khoang 7 - 8 . Vdi s g ho tro tieh ego eua ky thuat dien di va sac ky trao ddi ion hoan toan cd the phan tach va danh gia d u g e
dp tinh sach eao d c l p dp phan tip de tao ehe pham RNase dung djeh dat tdi 40 - 60 U/ml; ddng thdi ed the ddng khd dung djch nay de chuyen thanh dang bdt ddng khd tap d i i u kien thuan Igi cho viec san xuat che pham RNase diing lam thudc d i i u tri benh.
K i t qua xae djnh khdi l u g n g phan tir ciia RNase tru-ng l e h {Ranna rugulosa Wiegmann) a Viet Nam la 12 kDa eung tipgng d u o n g v d i nghien eu-u ciia eae tae gia khae: RNase ciia npc ran ^ va amphinase tir trirng l e h {Rana pipiens) i^-1.
Ket luan
Da Iga chpn d u g e d l l u kien thfch hgp de chilt tach RNase trung l e h (mdi t r u d n g dem PBS 0,1M pH 7,4, ii g ZT'C, 30 phut) de e h l tae dich chilt trirng l e h B (DCTEg) cd heat dp rieng RNase A_, la 3,27 ± 0 , 1 2 U/pg protein cao h g n cd y nghTa (p
< 0,05) so vdi mdi t r u d n g H^SO^ trong eung dieu kien (A„ = 2,72 ± 0 , 1 6 U/pg protein).
Tip DCTEg da thu d u g e e h l pham RNase cd heat dp 40 - 60 U/ml, khdi lu-gng phan tip 12 kDa va dat dp tinh khiet cap.
Tai lieu tham khao
1. Do Dire Giao (1999), "Thuat toan tim cay nhj nguyen t6i u-u vdl cac thong tin chira d cac la va trong cac dinh", Toan rdi rac, Nxb Dai hoc Qu6c gia Ha Noi, tr. 147-189.
2. Nguyin Van Thilt (2002), "Nghien ciru hoat tinh ribonucleolytic ciia npc ran ho mang", Tap chi Duac lieu. Tap 7 (s6 6/2002), tr. 181-185.
3. Biodiversity Committee, Chinese Academy(2013),
"Rana rugulosa Wiegmann" Catalogue of Life China, Annual Checklist.
4. Li S., Sheng J., Hu J. K. et al (2013),
"Ribonuclease 4 protects neuron degeneration by promoting angiogenesis, neurogenesis, and neuronal survival under stress", Angiogenesis, Apr, 16(2), 387- 404.
5. Woodclife Lake, N. J. (2008), "Preliminary results from onconase (R) phase lllb clinical trial", PRNewswire-First Call/Strativa Pharmaceuticals, May 29, 2008.
6. Sandeep K. Gupta, Brendan J. Haigh, etal (2013),
"The mammalian secreted RNases: Mechanisms of action in host defence". Innate Immun, Feb, 19(1), 86- 97.
7. Singh U. P., Ardelt W. et al (2007), "Enzymatic and structural characterisation of amphinase, a novel cytotoxic ribonuclease from Rana pipiens oocytes", Journal of Molecular Biology, 371, 93-111.
{Ngay nhan bai: 09/03/2016 - Ngay duyet dang: 25/04/2016)
