- 01 Thac Sl, bao ve nam 2005, vdi de tai lu$n van:
776 SINH HOC ca THEVA DINH HUONG UNG DUNG
HAA -^"r
35'C
40*C
Hinh 2. Anh h i r a n g cua nhi^t dp va pH len lectin lpc vCrng
Djch chiet thd (DCT) hgt lpc vung da duoc su- dyng de tinh che lectin bing s i c ky tren cpt trao doi ion DEAE 52 va tiep theo la cot be gel Sephadex G-75. Ket qua thu du(?c cho thay djch chiet thd (DCT) hat lpc vung sau khi qua cpt DEAE da cho mot dinh protein cd hoat tinh, dinh nay sau s i c ky lpc gel 6 budc tilp theo da cho ch4 pham lectin vdi dp tinh sach tang len g i n g i p ddi.
Khi kiem tra dp sach cua ch6 pham bing SDS-PAGE (hinh 1), cd the thiy tu rit nhieu protein a m i u thd (cpt 2), qua s i c ky trao doi ion edn lai bdn bang polypeptide (cot 3), sau do qua s i c ky Ipc gel chf cdn mpt bang duy nhat cd khdi lup'ng phan tu khoang 45 kDa (cpt 4), chi/'ng to che pham thu dup'c cuoi cung da hoan toan tinh sgch.
Nghien CLPU mpt s6 dac tinh cua lectin hat lpc virng
Anh hu^ng cua nhi$t 6Q: Hoat dp ciia lectin lpc vi^ng bj giam dan 6 nhung nhi^t dp cao hon nhiet dp
phdng (30, 35, 40 °C) trong vdng ti> 1- 72 gid xu ly. va mit hoan toan o 60 °C sau chua den 1h, nhung lai ben o khoang tiF 20° C den 4° C (Hinh 2). Do vay, nhiet dp tu 20 °C den 4 °C thich hop cho viec bao quan va tinh che lectin. Tinh ben nhiet ia mpt dgc diem chung cua cac lectin ngudn gdc thyc vat [2,3].
Anh huong cua pH: Do thj bieu didn tren hinh 2 cho thay lectin lpc vu^ng bieu hien hoat tinh cao 6 trong
vung pH g i n trung tinh va hoi kilm, cao nhit o pH 6,8 - 8,3. Hoat dp ciia lectin giam manh 6 cac pH lech khoi cac gia tri tren, dieu nay cho thiy pH cd anh huong Idn d i n hogt tinh sinh hpc ciia lectin.
T^c di^ng Ctc che cua saccharide: Tren thyc te. sy phan nhdm lectin thud'ng dya tren tuong tac dac hi^u
ciia chiing vd'i cac logi saccharide, do nhung u'ng dung trong nghien cifu sinh- y hoc cung nhu tiem nang dupe su dung trong cdng ngh# sinh hpc ciia lectin dua chii y l u tren dgc tinh nay [3,6]. Oe nghien eiru tuong tac dgc hi^u cira lectin, chiing tdi da su dung 12 logi saccharide khac nhau, trong dd ngoai cac logi dud'ng dan va dan xuit cdn cd mpt disaccharide (lactose) va mpt glycoprotein (IgG). Tren bang 2 la so lieu thu dupe khi xu* ly c h i pham lectin hat Ipe vung va dich chilt hat Ipe vC^-ng thd vd'i cae saccharide 6 cac nong dp khac nhau. Ket qua thu duoc cho thiy tinh dgc hi^u dudng ciia lectin Ipe vung la kha rpng. Ben canh fucose, galactose (Gal) va
disaccharide cd chipa Gal trong thanh phin la lactose ddu cd kha nang ifc che hogt dp lectin (a dgng chd pham hay trong dich
chiet thd) b ndng dp rat thip la 3,15 miyi.
IgG (cd chu'a Gal trong thanh phin oligosaccharide) cung cd tac dung len lectin o nong dp 31 mg/ml (vdi djch chilt thd) va 62 ng/ml (vai che pham).
Theo sy phan nhdm lectin dya tren tinh dgc hi^u dud'ng, cd the x i p lectin lpc vung vao nhdm II [3, 6]. Tren thyc t l , cd the su* dung cac nhdm lectin dac hi?u dudng khac nhau vao nghien ciPU nhgn dang te bao, phan biet
e^e vi sinh vgt, vi dy nhu vdi Helicobacter
pylori [4] va mpt sd loai vi khuin khac [1,5].
B a n g 1. Tac d u n g IPC c h e c u a c a c s a c c h a r i d e len
hoat d p l e c t i n hat lpc vCrng A-Cac mono- va disaccharide Galactose (Gal) Fucose Lactose Arabinose B- Glycoprotein IgG
NPng dp tdi thieu cdn gSy i>c c h i (mM) Dich chilt lectin tho C h i pham lectin
3,15 3.15 3,15 3,15 3,15 3,15 3.15 3.15 100 100 Nong do toi thieu cdn gay uc che (mg/m')
31 62 NhCrng saccharide khong co hoat tinh i>c ch4 cr npng do thii' NhCrng saccharide khong co hoat tinh i>c ch4 cr npng do thii'
nghiem cao nh^t (200 mM): glucose, glucosamine, N-acetyl D-
NHlfNG VAN DE NGHIEN CJfU CO BAN TRONG KHOA HOC Slf SONG 777
Bang 2. T i r a n g tac l e c t i n lpc vCrng-vi k h u a n
Salmonella W^u ATCC 9347 6/00 191 39 44 49 12-04
DCT 3+ 3+ 4+ 2+ 4+ 3+ 2^
CP 3+ 3+ 4+ 2+ 4+ 3+ 2+
(-) khdng cd phan ung ngung kit; (+) mice 66 gSy ngung kit;
Shigella; T: Staphyloccocus
Loai khac 104-04 6-04 105-04 94-04 E Sh T
1+ 2+ 3- 2+ - - -U 2+ 3- 2+ - - - U 2+ 3- 2+ - - -
DCT: dich chilt the CP: c h i phIm; E: E.coli; Sh:
Nghien cii'u tuong tac lectin- vi khuan: Chung tdi da t i l n hanh thii' nghiem tac dung gay ngung k i t ciia cac
m i u lectin vdi mpt sd vi khuan; Staphylo-eoceus, E. coli. Salmonella, Shigella. Pseudomonas aeruginosa va Proteus vulgaris n h i m de dinh logi chung. K i t qua thuc nghidm (bang 2) cho t h i y trong sd cac vi
khuan nghien cu-u, cac phan dogn ciia lectin lpc vCi-ng (tu dich chiet thd d i n c h i pham) d i u chi t u o n g tac vdi mpt loai duy n h i t la Salmonella. H a n nua, mifC dp gay ngung k i t ciia c h i pham so v o i m i u thd la khong doi; tuy t u n g ehiing ed the dgt mu-e tif y l u (1+) d i n taing binh (2+), manh (3+) va r i t mgnh (4+). Dieu n^y chirng to dp nhgy rat cao cua lectin: ehi can mot ham i u p n g lectin r i t t h i p o mau thd da cd kha nang gay n g u n g ket vi khuan.
Nhu vgy, ed the sip dyng che pham lectin ldc vO-ng de phan biet Salmonella vd'i cae lo^i vi khuan khac (qua phan ipng n g u n g k i t dac hieu), hon nua cdn cd the phan biet cac chiing Salmonella vd'i nhau ( d y a tren s y sai khac trong mu-c dp ngung k i t ) . K i t qua nghien cicu da gdp p h i n k h i n g dinh kha nang sif dyng lectin, dac biet la cac che pham tach t u ngudn thue vat phong phii eiia Viet nam. n h u mpt cdng cu huu ich de phat hi^n nhanh cae vi khuan cd t i m quan trpng trong sinh-y hpc,
TAI LI$U THAM KHAO