Phát hiện và sử dụng tính đặc hiệu của Lectin để nhận dạng một số vi khuẩn gây nhiễm độc thực phẩm

DAI HOC QUOC GIA HA NOI
TRI/CING
DAI HOC
KHOA
HOC
TI/NHIEN
DETAI
PHAT
HlfiN
VA
Sir DUNG
TINH DAC HIEU CUA LECTIN
¥)t
NHAN DANG MOT SO VI KHUAN
GAY
NHI£M
DOC
THUC
PHAM
MA SO: QG 04.12
CHU TRI DE
TAI:
TS.
Bui Phuomg Thuan
^^^
HOC
n(7?vV^~Tr
t>'r/ozzi
HA NOI - 2005
1.2.
Tinh

chat Iy hoa - sinh hoc cua lectin
1.2.1,
Cdu
tao phdn
tvr
cua lectin
Da so lectin co ban chit la glycoprotein, chi mpt so rat ft la protein dan
gian nhu ConA (chiet
tir dau
rua Canavalia ensiformis D.C) va lectin mam
liia
mi
[21].
Thanh
phSn
ducmg ciia lectin bao gom mannose, glucose, galactose, ngoai
ra con co sialic acid (la
dSn xu^ft
N-
acyl
ciia neuramic acid) thudng nam a phan
cuoi chu5i oligosaccharide cua cac glycoprotein hoa tan.
Thanh
phSn
protein gom mot hay nhieu
chu6i
polypeptide ket hgp lai nhd
lien ket cpng hoa tri. Lectin ciia m5i loai chiia thanh phan axit amin khac nhau,
nhung ph6 bien nha't la aspartic acid, serine, arginine va threonine. Cac axit
amin nay

chid'm tdi
30% thanh
phSn
phan tu lectin. Trong khi do, cac axit amin
chiia luu huynh nhu cysteine, methionine chi chiem mpt ti le thap hoac hau nhu
khong
CO
trong thanh
phSn
lectin.
Trong phan
tir
lectin con co cac ion kim loai, thudng gap nhat la
Mn^"^
va
Ca^"*".
Cac ion nay khong
true
tiep tham gia vao lien ket lectin-dudng, nhung lai
rat cin cho su tuong tac nay
vi
chiing anh hudng den ca'u hinh khong gian cua
phan
tir
lectin va trung tam lien ket dudng.
7.2.2.
Khdi luang phdn
tucua
lectin
Cac phirang phap sang lpc phan tu, dien di tren gel polyacrylamide, sieu

ly tam cho thay khoi lugng phan
tir
cua
letin
dao dpng
tir
vai nghin Da (8.500
Da d lectin rl cay gai
Uricaceat)
den vai tram nghin Da (700.000 Da d lectin
ciia sam bien chau A Tachypleus tridentatus)
[21].
Cho den nay, khoa hpc khong tim thay moi lien quan nao giua khoi lugng
phan tu va hoat tfnh
ciia
letin. Khdi Iugng phan
tir ciia
lectin khong mang tfnh
dac trung va khong phu thupc vao
miic
dp tien hoa cua loai hay ca the.
Do phan
tir
lectin co the dugc ca'u tao tu nhieu tieu don vi, nen khoi lugng
phan
tii
cua chiing phu thupc ra't nhieu vao ca'u tnic khong gian. Cac yeu to lam
thay ddi cau tnic khong gian cua lectin nhu: pH, dung dich ure nong dp cao,
guanidin clorit ddu co the anh hudng de'n khdi lugng phan
tir

lectin. Vf du ConA
d pH trung tfnh co cau tnic tetramer vdi khdi lugng phan
tir
khoang 102 000 Da,
nhung d pH < 6 cau tnic tetramer bi phan ly thanh cac dimer cd khdi lugng phan
tula 51 000
Da [21].
1.2.3. Tinh tan cua lectin
Lectin co kha nang hoa tan trong nudc, dac biet lectin ra't
dt
tan trong
dung moi la mudi loang, vi vay cac nha khoa hpc thudng
sii
dung cac dung dich
d6m
chiia mudi d nong dd sinh ly lam dung moi chie't xuat. Do ban chat la
protein nen lectin dl ke't tiia vdi aceton, alcohol d nhiet dp thap, mot sd mudi
trung tinh nong dp cao va mudi kim loai nang [26].
1.2.4. Khd ndng
tucmg
tdc vdi
du&ng
cua lectin
Kha nang tuong tac vdi dudng la mpt trong nhung
tinh
chat dien hinh ciia
lectin. Lectin cd the lien ket vdi ca phan tu dudng dan lan ca gdc dudng nam
tren be mat te bao. Cac loai dudng nhu glucose va glucosamine, galactose va
galactosamine, fucose, mannose tuong tac vdi nhieu loai lectin [21].
Cd ra't nhieu gia thuyet ve ban cha't su tuang tac giua lectin vdi gdc

dudng. Tuy nhien, cac nha khoa hpc deu thdng nha't cho rang su
bin
vung cua
phiic hgp lectin - dudng dugc duy tri nhd cac lien ket phan
cue
(lien ke't hydro
giua cac nhdm -OH ciia dudng vdi cac dau phan
cue
cua cac gdc axit amin tren
phan
tir
lectin) va lien ke't khdng phan
cue
hay lien ke't khong gian (lien ke't bang
luc Van der- Walls) [21]. Mankela da phat hien tha'y su khac biet trong ca'u tnic
khong gian d vi
tri
C3,
C4
cua cac loai dudng trong thanh phan cac loai lectin la
nguyen nhan chfnh dan den ai luc hoa hpc khac nhau cua chiing vdi cac loai
dudng [17].
Kha nang tuang tac dac hieu cua lectin - dudng da dugc
iing
dung nhieu
trong cac nghien
ciiu
te bao hpc, huyet hpc va miln dich hpc. Dac biet gan day,
nd cdn dugc
iing

dung de tinh che lectin. Nhieu glycoprotein cd chiic nang sinh
hpc da dugc tinh che bang phuang phap sac ky ai luc lectin.
7.2.5.
Cdc yeu
tddnh
hudng den
hogt
tinh lectin
Nhung ye'u td cd anh hudng de'n hoat tfnh gay ngung ket te bao (dac tfnh sinh
hpc quan trpng cua lectin) la pH, nhiet dp, cac loai dudng, enzyme
- Anh
hucmg
cua pH:
Do
sir
tuang tac cua lectin vdi cac hgp chat dudng (hoac cac thu the) la theo kieu
tuang tac ai luc hoa hpc, gia tri pH cd y nghia dac biet trong su tuang tac dd.
Ngoai ra, do lectin cd ban cha't la protein, hai gia tri pH can dugc luu y nhieu
nha't trong qua trinh tinh che lectin la pH dang dien va pH tdi thfch.
- Anh
hudns
cua nhiet do:
Nhi^t
dd cung tac dong
I6n ciu
tnic khong gian bac bdn cua phan
tir
lectin, lam
anh hudng
dd'n

ai luc
li6n
k^t
vdi dudng ciia cac trung tam hoat dpng va lam
thay doi hoat tfnh ciia lectin.
Da sd lectin hi ma't hoat tfnh d nhiet dp cao, khi lien ke't hoa tri cua phan
tu hi
diit
gay khien ca'u tnic khdng gian ciia chiing khdng ihi
phue
hdi lai (bi
bien tinh khong thuan nghich). O nhiet dp thap, hoat tfnh lectin bi giam, khi
tang din nhiet dd thi hoat tinh lectin cung ciing tang din den mpt gia tri
cue
dai
d nhiet dp tdi thich.
- Phdn img cua lectin vdi cdc logi dudng
Khi cd (cac) loai dudng dac hieu, trung tam hen ke't vdi saccharide cua lectin bi
phong be lam mit tac dung lien ke't vdi cac thu the tren bl mat te bao, lit dd
lam
mit kha nang gay ngimg ket ciia lectin.
- Tdc dung cua enzyme
Cac enzyme cd kha nang lam bie'n doi tfnh chat ciia mang te bao va lam thay doi
kha nang gay ngung ket te bao cua lectin.
Mpt sd enzyme nhu trypsin, papain cd tac dung lam tang hoat tfnh ciia
lectin. Khi hdng ciu dugc xii ly bang cac enzyme nay, mpt sd protein tren bl
mat mang te bao hdng cau se bi thuy phan lam bpc
Id
ro cac thu the cd ban chat
la glycoprotein, giiip cho lectin

gin
vao mang te bao hdng
cSu
dl dang han, do
vay se lam tang hoat tfnh ngung ket te bao hdng cau
ciia
lectin.
Ngugc lai mpt sd enzyme lai lam giam hoac mat hoat tfnh
ciia
lectin. Khi
xir
ly hong ciu bang galactose oxidase hoac mot sd glycosidase, cac gdc dudng
dac hieu vdi lectin bi phan huy, do dd hoat tfnh ngung ke't se bi giam.
- Anh hudng cua cdc ion kim logi:
Mot vai ion kim loai cung anh hudng den hoat tfnh
ciia
lectin. Tac dpng
ciia
chiing kha phiic tap khdng tuan theo quy luat nao: cd the lam giam hoac tang
hoat tfnh cua lectin.
1.3 Dac tinh sinh hoc va miln dich ciia lectin
7.3.7 Khd ndng gdy ngimg kei tebdo
Gay ngung ke't te bao la dac tfnh sinh hpc dien hinh
ciia
lectin, dua vao dd ma
ngudi ta phat hien lectin d cac ddi tugng nghien
ciiu,
cung nhu de phan biet
lectin vdi
nhOng

hgp chit sinh hoc khac cung cd kha nang tuang tac vdi dudng
nhu glycosidase, glycotranferase [26].
De cd the gay ngung ket
i6
bao, lectin phai tao ra nhilu cau ndi giua cac
t^
bao bi gin ket. Tuy nhien kha nang gay ngung ket khdng chi phu thupc dan
thuin vao sd lugng lectin, ma cdn phu thupc vao nhilu ye'u td nhu: dac tfnh phan
tiJ
ciia lectin, dac
tinh
mang tl bao (sd trung tam lien ket dudng, kfch thudc phan
tii,
sd lugng thu the, tfnh chit va tinh trang trao doi chit ciia mang), ndng dp te
bao,
pH, nhiet dp, chit kich thfch, chit kim ham, ion kim loai [41].
Hiu het cac lectin cd kha nang ngimg ket te bao hong ciu ciia ngudi
(trong dd sd lectin dac hieu vdi nhdm mau la rit ft), mpt sd cdn cd kha nang
ngung ket hong ciu ciia cac dpng vat khac nhu: thd,
cCai
Ngoai ra, ngudi ta
ciing
phat hien thiy lectin cdn cd the gay ngung ket ca
vi khuin, nim, sinh vat dan bao va cac bao quan (ty the). Chfnh vi vay lectin cd
vai trd quan trpng trong nghien
ciiu
khoa hpc va y dugc hpc.
7.3.2, Khd ndng kich thich vd kim hdm phdn bdo
Kich thfch phan bao cung la mpt dac tfnh quan trpng mang lai nhilu
iing

dung
thuc tiln cho lectin. Lectin
xam
nhap vao ca the cd tac dung nhu nhung khang
nguyen kich thich su phan chia cac te bao lympho (loai te bao dugc lectin kfch
thfch manh nhit), do dd giiip cho ca the tang cudng dap
iing
miln dich. Ngoai
ra, lectin cdn cd kha nang kfch thfch su phan chia cua nhilu loai te bao khac nhu
tebao
phdi ga ,.
Cho tdi nay, ngudi ta vin chua biet ro ca che kfch thfch phan bao
ciia
lectin. Cd y kie'n cho rang saccharide tren mang te bao la yeu td kim ham
sir
phan chia te bao, mpt khi bi lectin gin vao se bi mit tac dung, do dd lam tang
kha nang phan chia te bao. Tuy nhien gia thiet nay cling chua that thuye't
phue,
do nhilu lectin tuy lien ket dugc vdi cac phan tu saccharide tren bl mat cua
mang song lai khong cd kha nang kfch thfch phan bao. Nhung phat hien ve
glycoprotein mang te bao chi lien ket chpn lpc vdi nhirng lectin cd kha nang
kfch thfch phan bao (Centerlind va cpng
sir
- 1980) da din den kha nang tdn tai
cua nhirng thu the mang dac hieu vdi lectin kfch thfch phan bao [19].
Kha nang kfch thfch phan bao cua lectin dugc
sir
dung cd hieu qua trong
y hpc va miln dich hpc de nghien
ciiu

cac qua trinh bien doi sinh hoa cua te bao
tu trang thai nghi sang trang thai hoat dpng (cd tac dung phan chia te bao tfch
cue
va
gay
dap
iing
miln dich), cung nhu dl nghien
ciiu
su bit thudng ciia cac
nhilm sic the va cac htnh ly khac d ngudi.
Trong khi dd, mpt sd lectin lai cd kha nang
lic
che
sir
phan chia te bao,
nhu kim ham su phat triln va di can ciia te bao ung thu. Theo Brucke, dd la do
cac phan tu lectin da tac ddng den phan tu ADN khdi dpng cho su tong hgp
interferon
tir
te bao chu xam nhap vao te bao ung thu, do dd
lie
che su phat trien
ciia khdi
u [31].
Tren thuc te, su tuang tac giua lectin va cac glycoprotein dugc bieu hien
qua su tuang tac ciia cac globulin miln dich (IgG) vdi khang nguyen benh va
mpt sd
enzyme
7.3.3.

Ddp img mien dich
Trong nhung nam gin day, hang loat lectin dugc phat hien cd chiic nang sinh ly
va dap ling miln dich tren bl mat te bao ddng vat bac cao va ngudi. Selectin cd
kha nang lien
kl^t
vdi nhilu chit ket hgp dudng, cd vai trd
chii
ye'u trong ket dinh
cac te bao npi md thanh mach, kiem soat hien tugng viem va di can ung thu
[26]
L3.4.
Cdc quyei dinh khdng nguyen nhdn biei
Lectin cd the tham gia vao viec nhan bie't cac vi sinh vat ciing nhu nhan
biet cac te bao cua ca the. Kha nang nay cho phep giai thfch hang loat
sir
tuang
tac giiia cac te bao nhu
sir
ke't dfnh cua te bao vi khuan vao dpng vat, su lien ke't
ciia
vi khuan cd dinh dam d rl cua cay hp dau, su tiep can tinh triing vdi tning
Nhilu nha nghien
ciiu
da chi ra ring, tac dpng cua lectin ngoai sinh len
cac te bao dpng vat in vitro cd the gidng nhu tac dpng cua protein lien ket vdi
dudng, thudng xuit hien d bl mat
ciia
nhung te bao lan can [12].
Lectin cdn cd vai trd nhu la protein bao ve: bao ve thuc vat trong viec
chdng lai cac tac nhan tac dpng tu ben ngoai, ke't dfnh te bao va

gay
dpc ddi vdi
dpng vat [32].
Gidng nhu enzyme, khang nguyen va khang the, lectin cung la nhung
protein cd kha nang nhan bie't tinh vi cac hgp chit dudng tren bl mat te bao, do
vay Cling cd anh hudng de'n viec dilu chinh cac chiic nang sinh ly
ciia
te bao.
Lectin cdn cd
thi
ddng vai trd van chuyen chat du trii va lam nguyen lieu
du tru, lam chit trung gian lien ke't giira vi khuin va thuc vat trong hien tugng cd
dinh dam, vf du nhu lectin chiet
tir
cd ba la cd kha nang tuang tac vdi khang
10
nguyan
bl mat te bao vi khuin Rhizobium
trifollii,
lam ke't dfnh vi khuan nay vdi
t^baocuarlcaylll,
19,33].
1.4 Mot sd umg dung cua lectin
7.4.7 Trong huyei
hpc
Six
dung lectin de phan loai nhdm mau la
iing
dung sdm nhit, cho den nay vin
cdn dugc ap dung rpng rai. Phuang phap xac dinh nhdm mau bang lectin cho ket

qua nhanh, chinh xac ma khdng cin diing huyet thanh miu. Tuy nhien phuang
phap nay ddi hdi lectin phai cd dp tinh khiet va tfnh dac hieu cao.
1.4.2 Trong te bdo
hpc
Lectin dugc
sir
dung nhu mpt cdng cu huu hieu de nghien
cihi
ciu tnic mang te
bao va nhung bien doi tren bl mat mang te bao trong qua trinh biet hoa benh ly,
thdng qua su thay doi thanh phin cabonhydrat tren bl mat te bao.
1.4.3 Trong y hpc
Lectin dugc
sii
dung nhu mpt cdng cu chan doan cd hieu qua (dua vao
kha nang tac dung dac hieu len mpt sd loai vi sinh vat), ket hgp vdi cac xet
nghiem thdng thudng khac de nang cao gia tri chin doan benh.
True
khuan than
{Bacillus anthracis) dugc phat hien nhd xet nghiem ELLA vdi lectin dau tuang
{Glycin
max),
hay benh nhan nhilm ki sinh triing san mang {Schistosoma
mansoni) da dugc chan doan nhd
sii
dung lectin. Dua vao kha nang ai luc manh
vdi AFP, lectin
tir
dau lang va
tir

dau than dd da dugc
sii
dung de chain doan ung
thu bieu md te bao gan [24, 25].
Dua tren thuc te lectin thupc hp lan, hp thuy tien cd tac dung kim ham in
vitro su phat triln ciia virut HIV trong nudi cay te bao lympho T-
CD"*""
(do ke't
hgp duac vdi glycoprotein tren bl mat te bao the chd
ciia
HIV),
hay lectin
tir
hat
mit {Artocarpus heterophyllus) cd the kim ham hien tugng nhilm HIV va han
che kha nang mdc benh nhd tuang tac dac hieu vdi phan tu glycoprotein
CD^^
d
bl mat te bao T cua ngudi (la phan tu ke't hgp dac hieu vdi HIV trong pha dau
cua qua trinh nhilm benh), cd the du doan lectin se trd thanh mpt sinh phim cd
gia tri trong dilu tri va chan doan benh.
L4.4
Trong vi sinh vgt hpc
11
Do bl mat cua vi khuin mang cac gdc dudng, lectin cd the ket hgp vdi cac vi tri
lien
kft
tilm nang nay,
th^m
vao dd la tinh dac hieu khien chiing trd thanh cong

cu
hiru
ich trong
vi6c
nhan dang vi khuin
[11].
Do cd kha nang
li^n
ket vdi dudng va cac hgp chat chiia dudng (thanh
phin ciu tao quan trpng
lr€n
bl mat te bao), lectin cd tilm nang
iing
dung rat
ldn trong sinh- y hpc
[16,20,40].
Hiu het viec
sir
dung lectin trong nghien
ciiu
benh ly hen quan den vi
sinh vat
diu
dua tren ca sd phan
iing
ngung ket chpn lpc. Lectin WGA
(agglutinin mim
cay
lua mi) cd the dugc
sir

dung phat hien cac loai ciia chi
Neisseriaceae [32], hay
sir
dung WGA de phat hien Neisseriaceae
gonorrhoeae. Viec
six
dung lectin
kit
hgp vdi enzyme phii hgp da giiip giam thdi
gian thu nghiem ddi vdi Neisseriaceae gonorrhoeae, N.
meningitidis,
N.
lactamica vd Moraxell catarrhalis.
Mpt bang chiing khac vl tinh hiiu dung cua cac lectin dugc dua ra la kha
nang gay ngung ket chiing
Bacillus,
dac biet Bacillus
anthracis,
bdi vi rat khd
de phan biet vdi cac chiing B. cereas, B. mycoides vd B. thuringiensis. Phuang
phap gay ngung ke't de nhan ra
B.
anthracis la su dung cac lectin thu dugc
tir
dau nanh (SBA) va tii dc sen (HPA). Trong cac chung Bacillus, SBA chi ngung
ket B. anthracis va B. mycoides, nhung khdng tac dung len ddi vdi B.
thuringiensis. Han
nQa,
lectin cdn tac dpng len cac bao tu cua B. anthracis. Cac
ket qua tuang tu cung thu dugc ddi vdi loai B. thuringiensis [15, 31].

Viec nghien
ciiu
lua chpn lectin tuong tac dac hieu vdi cac loai vi khuan
cd the giiip ich trong viec dinh danh cac vi sinh vat nay ]14, 23].
1.5 Dai cuong ve mpt sd vi sinh vat gay ngo doc thuc pham
7.5.7 E.coli
Escherichia coli la vi khuan Gram am, hinh que, cd kfch thudc (1,1-1,5)
|im X
(2-6)
i^m
thupc hp Enterobacteriaceae la loai yem khf tuy tien. Da sd cac
vi khuin nay phat trien trong khoang nhiet dp
tir
5-12"
C, cd the ton tai rat lau
trong mdi trudng va nhay cam vdi viec thanh
triing,
ngoai ra cdn chiu dugc mdi
trudng cd axit va nhiet dp thap tdt han cac vi khuan khac [2].
Vi khuin dugc bao bpc bdi mpt he thdng mang va thanh te bao. He thdng
mang bao gdm mang ngoai va mang trong. Mang trong dugc cau tao gdm mpt
ldp lipid kep day khoang 8 nm cd chiia 40% phospholipide va 60% protein.
12
Mang ngoai la ciu tnic
Upid
kep ciia lipopolysacharide (LPS) va
phospholipid. LPS dugc ciu tao tii 3 thanh phin: lipid A, mpt chudi dudng phan
nhanh va khang nguyen O. Thanh phin cdn lai
ciia
mang ngoai la cac protein

[27].
Bao bpc he thdng mang ngoai la thanh te bao, dugc ciu tao nen tu
peptidoglycan
lien
ket ddng hoa tri vdi mang ngoai (peptidoglycan la he thdng
ciu tao ciia cac polymer do lien ket dong hoa tri ciia cac gdc dudng va cac don
vi peptide) [27]. He thdng cac polymer nim tren mang va vach te bao cd mang
cac gdc dudng la ca sd cho viec lien ket vdi cac loai lectin
[11].
E.coli
dMqc
phan loai theo tinh khang nguyen ciia chiing, chii ye'u la
khang nguyen loai O va H. Theo phan loai gin day ngudi ta chia E.coli thanh
nam nhdm (chii
y^u
dua
irtn
yeu td gay benh, trieu triing lam sang, dich tl hpc
va cac typ huyet thanh khac nhau): nhdm gay benh, nhdm sinh dpc td rupt,
nhdm
xam
nhap, nhdm gay dpc td mau va nhdm xam nhap theo dam [27, 35].
Do dugc truyln nhilm vao rupt chu yeu
tir
thuc phim, E. coli la nguyen
nhan quan trpng gay benh viem rupt, gay viem nhilm tao mu d nhilu nai [27].
E. coli cd kha nang khang lai cac loai khang sinh cd dien trudc day
thudng hay dung de dilu tri benh nhan tieu chay. Kha nang nay ciia chiing cd
thi
dugc hinh thanh do dot bien gen, di truyln qua cac the he, tie'p hgp hoac do

cac bien ddi thanh va mang te bao [4].
7.5.2 Proteus
Proteus la
true
khuin Gram am, cd long xung quanh than va cd kha nang
di dpng, la vi sinh vat hieu khf nhung cd the phat trien trong mdi trudng ky khf,
khdng len men lactose nhung cd kha nang len men mpt sd loai dudng khac, cd
sinh hai.
Proteus gdm bdn loai: P. vulgaris, P. mirabillis, P. morganii va P.
rettgeri. Tuy nhien mpt sd tac gia xep P. vulgaris, P. mirabillis vao chung mpt
loai P.
haiiseri
va dai dien dien hinh
ciia
Proteus la P. vulgaris [27].
Dua vao khang nguyen O va H, Proteus dugc chia thanh 100 typ huyet
thanh khac nhau. Dd la cac vi khuin ca hpi gay viem dudng tie't nieu, nhilm
khuin huyet [l]
7.5.3 Pseudomonas aeruginosa
13
True
khuin mii xanh
la
vi khuin gram
am
cd hinh thing hoac hai cong nhung
khdng
xoin,
hai diu trdn cd kfch thudc
tir

(0,5 - 1)
|im
x (1,5 - 5)
^m,
cd mgt
ldng duy nhat d mpt
cue,
cac
pili
(dai khoang 6nm) la noi tie'p nhan nhilu loai
phage va giiip cho vi khuin gin vao bl mat te bao
chii
[I]. Day la vi khuin hieu
khi tuyet ddi, cd the mpc tren mdi trudng thdng thudng trong khoang nhiet dp
tir
5 -
42°C,
nhiet dp tdi uu la
3TC
va pH tdi thich la 7,2 -7,5.
Tfnh chit dac tnmg ciia
true
khuin mu xanh la sinh sic td va chit tham,
vdi hai
loai
sic td chinh:
- Biocyalin cd mau xanh la cay, tan trong nudc va cloroform, khuech tan
tdt ra mdi trudng lam cho mdi trudng va khuan lac cd mau xanh .
- Bioverdins la
loai

sic td huynh quang, phat miu xanh khi chieu tia cue
tfm cd budc sdng 400 nm.
Pseudomonas aeruginosa la
loai
vi khuin gay benh cd dilu kien, chiing
xam nhap
til
mdi trudng ben ngoai vao ca the qua cac vet thuang hoac khi ca the
bi suy giam miln dich hay bi mic benh man tfnh. Nhiing ngudi diing
corticoid
keo dai, khang sinh hoac chit chdng ung thu thi dl mdc benh nhilm triing npi
sinh hoac ngoai sinh do true
khuin
mu
xanh.
True
khuan mu xanh khang lai
nhilu loai khang sinh thdng dung (penixilin, ampixilin, cloramphenicol va
tetracyclin), d nudc ta cd khoang 407o sd chiing
true
khuin mu xanh khang
dugc gentamycin
[ 1
].
1.5.4 Samonella
Samonella la vi khudn Gram (-)
hinh
que cd nhieu long, kich thudc trung
binh 3 x 0,5
j^m,

sdng hieu ki khi tuy tien, phat trien tren mdi trudng thdng
thudng. Cho de'n nay da cd han 1800 chung typ huyet thanh thupc loai
Samonella
[6,
28].
Thanh te bao vi khuin Samonella dugc ciu tao
tir
peptidoglycan va
techoic acid. Peptidoglycan la loai polyme xdp, khdng tan, dugc tao nen
tir
aceiylglucosamine,
N-acetymuramic acid va tetrapeptide. Cac techoic acid la
cac polyme lien ket vdi peptidoglycan va mang te bao chat. Bao ngoai thanh te
bao la mpt ldp lipid kep day khoang 3-7 nm dugc cau tao tir photpholipid,
lipoprotein,
liposaccharide (LPS) va cac protein [27, 28].
Samonella cd mat d khdp mpi nai tren ca the ngudi, tap trung chu yeu d
dng tieu hoa va gay ra cac benh truyen nhilm nguy hilm nhu nhilm khuan
dudng tieu hoa, viem dudng rupt, nhilm triing mau va thuang han [3, 28].
14
Samonella cd hai loai khang nguyen chinh: khang nguyen than O (cd ban
chit la lipopolysaccharide (LPS) d mang ngoai, chiu dugc nhiet, khang con, axit,
ddng vai trd nhu mdt npi dpc td) va khang nguyen long H (cd ban chat la protein
n^n
dl bi bit hoat bdi
nhidt
dp, cdn va axit). O mpt vai chiing cd them khang
nguyen bl mat tham gia vao viec bao ve vi khuan
[1,
28],

Hien nay ti
16
Samonella khang thudc ngay cang gia tang, da cd tdi 40%
chiing dugc phan lap (nam 1998) khang lai ampicillin va 62% khang lai
chloramphenicol
[1,7].
1.5.5
Shigella
Shigella la
true
khuin gram am, manh, dai tir 1-3
^im,
khi mdi nudi ciy cd
dang
true
ciu khuin. Shigella khdng cd long, khdng cd vd va khdng sinh nha
bao,
la vi khuan hieu khi tuy tien nhung phat trien tdt trong dilu kien hieu khf
[1].
Thanh te bao Shigella cung bao gdm cac thanh phin gidng nhu
Samonella: peptidoglycan chi chiem mpt ti le nhd (5-10%), cac tetrapeptide tren
mdi chudi peptidoglycan lien ke't cheo vdi cac tetrapeptide khac, ngoai ra cdn
lien ket vdi techoic acid [27].
Mang ngoai ciia vi khuin la ldp lipid kep cau tao tii ba thanh phan chfnh:
khang nguyen O, lipid A va protein ca chat. Trong dd thanh phdn khang nguyen
O chfnh la cac polysacharide (la vi tri lien ket lectin tilm nang trong phan
iing
ngung ket vi khuan) [27].
Tit ca cac Shigella
diu

cd khang nguyen than O, mpt sd cd khang nguyen
K va
diu
khdng cd khang nguyen H. Dua tren tfnh dac hieu khang nguyen than
O
va mpt sd tfnh chit sinh hoa, ngudi ta chia cac Shigella thanh 4 nhdm: A {S.
dysenteriae), B
{S.flexneri),
C
{S.
bodii) va D ( 5. sonnei) [1]
Shigella la tac nhan gay benh ly
true
khuan. True khuan lay theo con
dudng sinh hoat vao dudng tieu hoa, gay ra xung huye't, xuat huyet, tao ra nhung
d loet, mang hoai tir va tac dpng len thin kinh giao cam, gay ra co that va tang
nhu ddng rupt
Shigella cd kha nang khang lai cac khang sinh pho bien nhu
streptomycin, ampicillin, tetracylin Hau het cac chiing S.flexneri nhay cam
vdi
mecillin
va ciprofloxain [4].
1.5.6 Staphylococcus
15
Tu ciu vang {Staphylococcus aureus) la nhung ciu khuin, cd dudng kfnh tir 0,8
- 1
|im,
tap hgp thanh hinh
chiim
nho, bit miu gram duong, khdng cd long,

khdng nha bao va khdng vd. Cac
loai
thudc chi Staphylococci
diu
la vi khuin
yim
khf tuy tien.
Tu ciu vang cd kha nang dl khang vdi nhiet dp va hoa chat cao han cac vi
khuin khdng nha bao khac, bi diet d nhiet dp
80"C
trong mdt gid, tuy nhien kha
nang dl khang vdi nhiet dp thudng phu thudc vao kha nang thich
iing
vdi nhiet
dd tdi da ma vi khuin cd the phat trien
[1,6].
Ciu tnic thanh te bao ciia vi khuin cd cac nhdm chit nhu polysaccharide
va glycoprotein (cac nhdm chit mang gdc dudng cd
thi la
vi tri lien ket vdi
lectin trong phan
iing
ngung ket) [27].
Nhilm vao ngudi khi an phai thuc phim khdng dugc bao quan tdt, loai vi
khudn nay gay ndn
mira
nhilu va dau co thit viing bung, cd the gay tieu chay
hay dau diu [1]. Da sd tu ciu vang khang lai penicillin G, mdt sd cdn khang
dugc methicillin
[1,4],

7.5.7 Phuong phdp dinh dgng vi sinh vdt
Cac quy trinh phan tich va xac dinh vi sinh vat dugc xay dung tir nhimg
nam 80 cua the ky 19 va da dat dugc nhimg thanh qua nhit dinh.
Diu tien la cac phuang phap truyln thdng nhu quan sat bing kinh hien vi,
thir
nghiem sinh hoa, sir dung cac mdi trudng chpn lpc, cac bd kit sinh hoa va
cac he thdng dinh dang tu dpng trong viec xac dinh vi sinh vat [2, 10].
Tiep de'n la cac phuang phap khdng truyln thdng (nhung cai tien dua tren
nhung phuang phap truyln thdng) nhdm tang hieu qua trong viec kiem nghiem.
Hiu het cac phuang phap thii nhanh tu dpng diing trong cdng nghiep hien nay
diu
dua tren cac phuang phap phan tich miln dich (ELISA, LA, IFA, IMS),
diing trd khang, phan tich axit nucleic (miu dd, PCR) hoac cac ky thuat phat
quang sinh hpc khac. Cac phuang phap nay cd the sir dung ddc lap hoac phdi ket
hgp vdi nhau, vi du ket hgp phuang phap phan tfch nhanh IMS vdi ky thuat
ELISA dl xac dinh vi khuin [6, 10].
Gin day cac nha khoa hpc da
sir
dung lectin trong viec xac dinh va phan
biet cac chiing vi khuin dua tren kha nang tuang tac
ciia
lectin vdi cac gdc
dudng [9, 14, 19, 22]. Do hiu
hit
cac vi sinh vat
diu
cd cac gdc dudng tren bl
mat te bao, day cd
thi
la vi trf phan

iing
tilm nang
ciia
cac lectin [34]. Tren ca
sd dd, cd
thi
sir dung cac miu lectin tach tir sinh vat trong thien nhien nhu
16
nhihig cdng cu trong viec dinh dang cac chiing vi sinh vat, hay phan biet cac loai
cd type
huyl^t
thanh khac nhau nhu: sir dung lectin WGA (agglutinin mim
liia
mi) phat hien cac chiing vi khuin thudc chi Neisseriaceae gay ra cac benh viem
mang nao, tiet nieu, sinh
due
[24], gay ngung ket va xac dinh dugc cac chung
cua nhdm Campylobacter jejuni vd Campylobacter coli [44], sir dung mdt sd
ddi tugng lectin trong viec xac dinh tu ciu vang va tu ciu trang[9,
18,
22]
17
t^A'HOC
QUOC GIA HA
NOl
TRUNG TA^-HONG
TIN
THl.r
v/iPN
P/

/5~2i
CHl/ONG
2 -
NGUYfiN
LIEU VA PHlTONG PHAP
2.1 Nguyen
lieu
2.2.7 Ddi
tuptng
nghien cim
Nguyen lieu de nghien
ciru
la cac loai hat, qua, la va than thuc vat mpc ngoai tu
nhien dugc thu hai tii cac tai cac
tinh:
Ha Ndi, Ha Tay, Nam Dinh, Hai Phdng,
Thanh Hoa, Quang Ninh Mpl sd miu dugc mua tai cac cira hang thudc Ddng
Y d Ha Ndi. Cac miu dugc dinh
t^n
theo su phan loai
ciia
Dd Tat Lgi, Vd Van
Chi va Nguyin Van Khdi.
Hong ciu cac nhdm mau do Vien Huyet hpc va Truyln mau Trung uang
cung cip.
Cac chiing vi sinh vat dugc phan lap
xix
cac benh nhan ngd ddc thuc phim
thu thap
tir

Vien ve sinh dich tl Trung uang va cac chung vi sinh chuin qudc te
do Trung tam bao tdn gidng vi sinh vat cung cip.
2.
7. 2 Hod chdt vd thiei bi
Cac hda chit, dung dich dem sinh phim sii dung trong nghien
ciiu
dugc mua tir
cac hang Sigma, LKB, Merk
diu
cd dp tinh khiet cao.
Cac thiet bi chinh diing trong thuc nghiem bao gdm:
- May ly tam MR 18, Jouan - Phap.
- May ly tam lanh eppendorf
5417R
-
Diic
- May do quang pho Shimazu UV-200 - Nhat ban,
- May do pH ATI- ORION - Bekman-My.
2.2 Phuomg phap nghien cuu
2.2.7 Tdch chiei cdc mdu lectin tho
Lectin
tir
cac miu thi nghiem dugc chiet xuat bing dung dich dem PBS pH
7,4 (hoac Tris -
HCI
(TBS) 20 mM pH 8,3) vdi ti le 1/10
(kl/tt)
(vdi nguyen lieu
tuai) va 1/20 (kl/tt) (vdi nguyen lieu d dang bdt khd), khuay ttr 30 phiit, ly tam
8000v/phut trong 15 phiit d nhiet dp

4"C,
loai can, thu dich trong.
18
2.2.2
Xdc dinh hogt dp lectin
bdng
phdn
img
ngung kei hong cdu
[211
Hoat dp ngung ket hong ciu (viet tit la HAA) la nghich dao cua dp pha
loang ldn nhit cua dich lectin cdn cd kha nang gay ngung ket toan bd lugng
hong ciu.
Cach tien hanh:
Hdng ciu cac nhdm mau da dugc chdng ddng bdng ACD (acid citric
detroza) (do Vien Huye't hpc va Truyln mau Trung uong cung cap), dugc rira (3
lin) bing mudi sinh ly vdi ti
le
1/20
(v/v), ly tam 1400v/phut trong 10 phut va
pha loang den nong dd 2% v/v.
Cho vao mdi gieng cua ban nhua day nhpn microliter 50
^il
dem. Cho 50
\x\
miu lectin vao gieng diu tien, trdn
diu,
lay 1/2 (50
\x\)
cho sang gieng

thii
hai,
trpn
diu,
lam tuang tu vdi cac gie'ng tie'p theo cho de'n het, bd di
50|il
trong
gilng cudi cung sau khi trpn.
Cho vao mdi gie'ng 50
\i\
hdng ciu da hda loang, de ban nhua d
37"
C,
sau
30 phiit dpc ket qua.
2.23 Tuong tdc
giira
lectin vd
tebdo
vi khudn [30].
Cac vi khuan thir nghiem dugc ciy truyln tir dng gidng qua mdi trudng
thach thudng, de qua dem trong dilu kien thudng.
Tir
dng thach cho 5 ml dung
dich mudi sinh ly, diing que cay thu sinh khdi, dich vi khuan dugc bat hoat bing
dung dich formaldehide 0,5%. Tiep den, tien hanh
rira
te bao bdng mudi sinh ly,
ly tam thu tua, hoa tan tua trong dung dich dem PBS pH 7,4, chinh dp due cua
dich huyin phu d budc sdng 540 nm den OD = 1 (tuang

iing
vdi
10'"
te
bao/lml).
Trdn 50
|-il
dich te bao vi khudn vdi 50
|il
dung dich lectin tren cac gieng
cua ban
thir
Boener (vdi gieng ddi chiing thay lectin bdng dem PBS pH 7,4), ldc
nhe,
sau 30 phiit quan sat ban
thir
tren hop guang.
Ket qua ngung ke't se la duang tfnh neu thay xuit hien cac khdi ket tu ciia
te bao vi khuin (thanh timg mang ldn hoac nhd), hoac te bao bi phan giai.
Miic
dp ngung ket phu thupc vao hoat dp cua lectin (4+; phan
iing
duang tfnh vdi
khdi tu ldn, 3+: phan
iing
duang tfnh vdi khdi tu kfch ca trung binh,
2+:
phan
ling duang tfnh vdi khdi tu nhd,
l-i-

ngung ket yeu, - : khdng cd ngung ket). Vdi
19
kSt
qua
am
tinh, cac
i€
bao vi khuin vin d dang huyin phii hoac ldng
diu
d giira
giiing
thiir.
Trong nghien cuu cdn sii dung cac phuang phap tinh sach lectin bdng sdc
ky tren cdt trao doi ion DEAE 52 va cpt Ipc gel Sephadex G-75; xac dinh ham
lugng protein bing phuang phap do quang pho va ky thuat Bradford; xac dinh
dd sach ciia che phim lectin bing
di6n
di bien tfnh tren gel polyacrylamide
(SDS-PAGE); nghien curu anh hudng
ciia
cac ye'u td: nhiet dp, pH va tac dung irc
ch^
cua cac hgp chit dudng
ien
hoat dp cua lectin.
2.2.4 Cdc quy trinh
chiei
xudt vd tinh che lectin
20
Mdu hat

lpc
vimg
Hat ldc vimg dugc nghien nho, ngam
trong dem Tris-HCl 0,01 M pH 8,3,
khuiy tir trong 3 gid. Ly tam 8000 v/phut d
4**C
vdi thdi gian
20'.
Dich chiet tho
Nap dich chiet thd len cdt sdc ky trao doi ion
DEAE sephadex A-50, tao gradient
ning
dp
NaCl de ddy cac protein gdn tren cpt. Thu
cac phan doan cd hoat do lectin cao nhat.
Dinh cd hoat tfnh
LVl
Nap dinh LVl len cpt sdc ky lpc gel
Sephadex G-75
Thu che pham
Dien di tren gel polyacryamide SDS-PAGE
12,5
%.
Kilm tra dp tinh sach
ciia che pham
jtiuTafagJirtntrffiPrag.atJcitr.'ijgi^Ciz.'c?-
So
do 1 - Quy trinh chiet xuat va tinh che lectin ldc
vimg
21

Mdu hat dao bien
Nghien hat dao bien thanh bpt min.
Ngam va khuiy tii trong dem PBS pH 7,4
trong 3 gid.
Ly tam 8000
v/phiit
6
4*'C
vdi thdi gian
20*
Nap dich chiet thd len cot sdc ky trao d6i ion
DEAE Sephadex A-50, tao gradient nong dp
NaQ
de day cac protein gdn tren cpt. Thu
cac phan doan cd hoat do lectin cao nhat.
Dinh cd hoat tfnh
DBl
Nap dinh DBl len cot sdc ky lpc gel
Sephadex G-75
Thu che pham (CP)
i
Dien di bien tfnh tren gel polyacryamide
(SDS-PAGE) 12,5
%.
Kilm tra dp tinh sach
cua che pham
So
do 2 - Quy trinh chiet xuat va tinh che lectin hat dao bien
22
Dich chiet

tebao (DC
2)
Ket tiia bdng amoni sulfate
a
4°C.
Ly tam 5000 v/p de thu protein ke't
tiia.
Hoa ket tua bdng dem Tris-
HCI
15 mM pH 8.0.
Thdm tfch trong ciing loai
dem
d
4"C
qua dem.
Dich sau ket
tiia
(DSD
Chay sdc ky loc gel G-75 d
4°C
sir dung dem Tris -
HCI 15
mM pH 8.0 cd chiia
ion 10 mM
Ca^^
Dinh 2 loc gel G-75 (LG 2)
Chay sdc ky trao
d6i
ion DEAE 52 d
4"C,

sir dung gradient nong dp NaCl de day protein
lectin khdi cdt
Che
pham lectin (CP)
So do
3 - Quy trinh chiet xuat va tinh che lectin hemocyte sam bien
23
Chuang
3-
Ket qua va ban luan
3.1 Khao sat hoat tinh gay ngung ket hong cau
ngucri
cua mot sd loai thuc
vat Viet Nam
Chung tdi da tien hanh khao sat hoat tinh gay ngung ket hdng ciu ciia han 100
miu thuc vat thudc 50 loai d 28 hp khac nhau.
Trong tong sd 50
loai
khao sat (bang 1), 32 loai cd hoat tinh ngung ket
hdng ciu vdi
miic
dd khac nhau, tap trung d cac hp; Arecaceae, Brassicaceae,
Cucurbitaceae, Fabaceae, Dliciuacea,
Lecythidaceae,
Liliaceae, Meliaceae,
Millettia,
Momordica, Moraceae,
Papihonoidea,
Sapindaceae, Solanaceae,
chi^m

mdt
ti
le kha cao la 64%.
Hiu het cac lectin cd hoat
tinh
ngung ket hong ciu
diu
khdng dac hieu
nhdm mau, trir mot vai ddi tugng nhu: dau ngu {Phaseolus lunatus L.J, hat bang
{Terminalia catappa L.) dac hieu vdi nhdm mau A, dac biet
la
miu hat vai
{Litchi sinesis) chi gay ngung ke't hdng ciu dac hieu nhdm mau
O.
Trong sd cac miu khao sat cd su phan bd khac nhau va su thay doi hoat
tinh ciia lectin d cac bp phan nhu; than, la, qua xanh, qua chin, hat non, hat gia.
Su khac biet nay cd the lien quan de'n qua trinh sinh tdng hgp lectin, su phat
triln trong
tiitig
giai doan phat trien cua cay hay do nhirng bie'n doi sinh hoa
khac trong cay [12].
Trong cac miu nghien
ciiu,
cac lectin cd hoat tinh gay ngung ket hdng ciu
thudng tap trung d hat va qua (nhu miu hat dao bien Canavalia maritima), dilu
nay cd the giai thich la cac lectin dugc sinh long hgp chii ye'u d hat cd the lien
quan de'n chiic nang du trir ciia cay. Trong khi dd, d miu hoe bui {Sophora
tomentosa L.), hoat tfnh gay ngung ket lai tap trung d hoa va vd, cd the do
lectin dugc tong hcfp d cac bd phan nay tham gia vao chiic nang bao ve
ciia

cay
[32].
Ke't qua dilu tra cho thay mpt sd loai cd hoat tinh gay ngung ket hdng ciu
rit cao nhu ldc
vtmg
{Barringtonia acutagula
L.),
dao bien {Canavalia
maritima),
cam thao day {Abrus precatorius
L)
Nhixng
miu nay da dugc
chiing tdi tap trung nghien ciru sau han.
24
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fS
Amonutu

aromaticum
1)
u
o
^-
ci:
Zin
o
W-,
3*2 Phat hidn va phan
I$p
mot so
loai
vi khuan gay nhilm doc thuc pham
Benh
ph^m
tur cac
b^nh
nh^n
bi
ng6 dpc
thuc pham, cac nguon thuc pham hay
gia Stic
gay
b^nh
da duac thu thap va bao quan.
Tijr
do. cac
miu
vi

khu^n gay
d6c
da duac phan lap tai
Vi6n
ve sinh dich td trung uang va Trung tam bao ton
giong vi sinh vat, duac nuoi ca'y va xu ly tru6c khi sir dung cho nghien
cun.
Cac chung chu yeu bao gom:
+ 9 chung tu
cSu
vang Staphylococcus aureus (cac miu 25, 64,
141,
143
duac phan lap
tijf
phan; 2-03, 2-04 duac phan lap tir dich thi
hSu
cua ngucfi
b6nh,
con 25923 la chung
qu6c
i€).
+ 5 chung tu
cSu
trang Staphylococcus epidermidis
(5-03,
1-04 phan lap
tijr
thit gia
cSm

gay
d6c
thuc phdm; 1-03
tiT
thjt gia
siic
gay benh, con 5-02 va 12-
04 phan lap
tilr thtic
an).
+ 5 chung
E.
coli
(3-04,13-04,
24-04 duac phan lap tir phan cua ngucri benh,
25922 la chung
chu^
cua tap doan giong quoc te).
+ 3 chung Salmonella (ca ba mSu A, B, D
diu
duac phan lap tir phan cua
ngucri
b6nh).
+ 3 chung Shigella (213, 474 phan lap tir phan, con 259 phan lap tir mau
ngucri benh).
Ngoai ra con co sau chung chuan cua cac tap doan giong qudc te do trung
tam bao tang gidng chuan vi sinh vat - Trung tam cong nghe Sinh hoc cung cap
bao gom: Pseudomonas
aeruginosa,
Proteus,

vulgaris (BV 108), E.coli (ATCC
9946 viet tat la:
E,),
Salmonella (ATCC 9347 viet tat la
S,),
Shigella (ATCC
7896 viet tat la: Sh,), Staphylococcus (ATCC 1222 viet tdt la:
Ti).
3.3 Phat hien cac lectin co kha nang nhan biet mot sd chiing vi khuan gay
doc thuc pham
Ket qua thu nghiem tac dung gay ngung ket cua 20
miu
lectin voi 25 chung
vi khudn da bat hoat duac trinh bay tren cac bang 2 va 3.
Trong sd nay, cac
mSu
dich huyet sam, dua hau, vai khong gay ngung ket
ba't ky mot chiing vi khuan nao (mac
dii
vln gay ngung ket hdng clu), do vay
khong
CO
tac dung trong phan loai.
25
rr
1
2
3
4
5

6
7
8
9
10
11
12
13
14
15
16
17
18
19
20(
Miu
Hoa hoi
Lac
Dau ngu
Dau
dai
Cam thao
day
CP lectin CTD
Qu6
Bura
Roi
Qua
lau
Mudp

ding
Xo^i
D^n
tia
Hoa hoe
H6ng
xiem
Ca chua
Cudau
Khoai tay
Hemocyte sam
P
lecti
n sam
%
miu
gnk
Bang 2- Tuong tac
lectin-
tucau
Staph,
aureus
15923
2-04
2+
2+
+
2+
-
+

2+
+
-
+
+
-
2+
+
-
+/-
55
3+
+
-
+
-
+
2+
2+
+
2+
2+
-
-
+
-
-
50
25
3+

2+
-
-
+
+
4+
2+
+
+
+
+
-
+
-
-
55
64 141
3+ 2+
3+ 2+
+
2+ +
+ +
+ +
2+ -
2+ +
- 3+
+ +
+ +
-
+

- 2+
-
-
50 55
143
3+
3+
-
-
+
+
2+
2+
2+
2+
-
-
2+
-
-
-
45
150
3+
2+
-
-
+
-
+

3+
-
+
+
2+
-
-
-
40
152
2+
+
-
-
-
-
+
2+

+
-
-
3+
-
-
30
2-03
3-¥
3+
+

-1-
-
-
2-1-
3-1-
-t-
-
-
-
+
-
40
Staph,
epidermidis
5-02
2-1-
2+
-
-
+
-
-
-
-
3-1-
-
-1-
-
-
3+

30
1-03
5-03
3-J-
-
-
-
-
-1-
+
3+
-
2+
-
-
-
-
-
25
-»-
-
-
-
-
-
H-
2+
-
+
-

-
2+
-
-
-
25
1-04
12-0^
2-1-
-»-
5-1-
-
-
-
-1-
-»-
+
+
+
-
+
+
-
2+
-1-
_
-
-
-
35 25

Ghi chu : (-) khong co phan
iJng ngixng
ket;
(-»-)
muc do
gay
ngung ket (gnk), CP: che phim.
Nghien
cun
da cho tha'y kha nang cua mot sd lectin trong viec phan biet cac
chung tu clu trlng va tu cau vang qua viec chi gay ngung ket dac hieu ddi vai
mot trong hai loai (Bang 2). Co 7 mlulectin tac dung ddi vdi tu clu vang la
lectin tach tir hat dau ngu (3), dau dai (sd 4), qua lau (sd 10), xoai (sd 12), den
tia (13), hdng xiem (sd 15) va cu khoai tay (sd 18). Nguac lai lectin tach
tijr
hat
mudp
ding
(sd 11), hoa hoe (sd 14) va qua ca chua (16) chi gay ngung ket vdi
cac chimg tu clu trlng.
Tuofng
tu nhu vay,
miu
dong vat duy nhat dugfc thir
nghiem la lectin sam bien a dang che pham (sd 20- tinh sach
bing sic
ky loc gel
va trao doi ion) cung gay ngung ket rat manh chung tu clu trlng S. epidermidis
5-02 ma khong cd tac dung len bat ky chung tu clu vang nao. Nhu vay, tat ca
nhimg lectin nay deu co the duac sir dung de phan biet giua hai loai tu clu. Ve

tong the, sd luang cac chung tu clu vang bi ngung ket duai tac dung cua lectin
la cao han (45- 55%) so vdi cac chiing tu clu trlng (25- 35%).
Ddng thdi, mot sd lectin cdn cd kha nang phan biet cac vi khuan trong ciing
mot loai. Vi du, lectin tir hoa hoe cd
miic
do gay ngung ket khac nhau tuy thuoc
26
ttog
chung tu clu trlng (manh nhit vdi 5-02,
y^u
han vdi 1-03, sau dd vdi 1-
04),
tuang tu nhu vay la lectin tii qua ca chua (sd 16). Trudng hop lectin tir hat
lac (sd 2) tuy cung tac ddng len
hiu h6't
cac loai vi khuan thu nghiem, nhung vdi
su chtnh
Itch
ra't ldn ve
miic
dd:
Staph,
epidermidis 1-04 bi ngung ke't dac biet
manh so vdi cac chung khac,
vi
vay cd the duac sir dung de nhan bie't chung tu
clu trlng gay doc thuc
phim
nay.
Thdng thudng, da sd cac phdng thi nghiem sir dung catalase va xet

nghiem ddng tu
huyft
thanh cung vdi phan
iing
nhudm Gram de phan biet tu clu
vang vdi tu clu trlng. Nhung ket qua thu
dugc
cua nghien
ciiu
cho tha'y cd
th^
su dung lectin vdi mdt quy
trinh
don gian va da tdn kem han de lam viec nay
trong thdi gian 5 phiit: ket hgp mot hoac mot vai lectin chi gay ngung ke't tu clu
vang (trong sd 7 loai cd tinh cha't nay nhu lectin xoai, dau dai, dau ngu, khoai
tay ) vdi lectin lit te bao sam (chi gay ngung
kft
tu clu trlng).
Tur
k^t
qua cua bang 3, trong sd nhiing lectin cd kha nang nhan biet su sai
khac giua cac loai vi khuin,
b^n
canh
miu
lectin tir dau ngu (da de cap d tren),
lectin hoa hdi
(miu
sd 1) chi gay ngung ke't duy nhat cac chung tu clu ma

khong tac dung
Itn
cac vi khuin G(-) la E.
coli.
Salmonella hay Shigella
(miu
khoai tay (18) va che
phim
lectin cam thao day (6)
cQng
cd tac dung
gin
tuang
tu).
Dieu nay cho tha'y nhung lectin nay khong cd cac vi tri lien ket vdi
phin
carbohydrate ciia cac lipopolysaccharide (LPS) cd tren thanh te bao vi khuin G
(-) va cd the
diing
de nhan biet loai vi khuan nay.
Lectin cd trong hat mudp
ding (11)
cd tac dung r5 ret len E. coli (vdi
miic
do manh ye'u khac nhau tuy thupc timg chiing) ma
hiu
nhu khong cd (hoac neu
cd chi la phan
iing
ye'u) vdi mot vai chiing thuoc cac loai khac. Trong khi dd,

dich chiet tho hemocyte cua sam (sd 19) gay ngung ket manh
nhit
chung 0127,
tiep theo la chung 24- 04. Cdn lectin tir hat hdng xiem thi chi gay ngung ket
chung 1027. Ba loai lectin nay do vay cd the sir dung de phan biet cac chiing E.
coli thir nghiem.
Ddi vdi Salmonella, cac lectin tir hoa hoe (sd 14) cd tac dung kha manh
vdi chung A, cu dau (sd 17) tac dong len chung D, cdn dau dai cd lien ket vdi
cac chung B, D. Ngugc lai, lectin tir hat cam thao day (du la d dang dich chiet
thd hay che pham) va lectin tir hat
bila
deu chi nhan bie't cac chung E. coli va
Shigella ma hoan toan khong cd tac dung len Salmonella. Do vay, cd the sir
dung phdi hgp cac lectin nay de phan biet Salmonella vdi nhirng loai vi khuin
kia.
27