Nghiên cứu giải pháp công nghệ FPGA cho các ứng dụng truyền thông

'M'"

DAI HOC QUOC GIA HA N Q I

Ten de tai
NGHIEN CLTU KY THUAT GAN KHANG N G U Y E N / K H A N G THE LEN BE
MAT HAT LATEX KICH THU6C NANO VA LTNG DUNG TRONG CHAN
DOAN NHANH BENH 0 NGUdi VA GIA SUC
(Bao cao tong hgp ket qua thuc hien
De tai nghien cuu Khoa hoc cap DHQGHN)

Mas6:QC.06 27
Chu nhiem de tai: TS. Chu Hoang Ha

DAI HOC QUOC GIA HA NO!
TPUNG TAM THONG TIN THU VIEN

000G000OO5'5

Ha N6i-2007


MUC LUC
3. Danh sach nhung nguai thuc hien

1

4. Danh muc cac bang so lieu

1

5. Danh muc cac hinh

1

6. Tom tat cac ket qua nghien cuu chinh ciia de tai

3

6.1. Ket qua khoa hoc

3

6.2. Ket qua phuc vu thuc te

3

6.3. Ket qua dao tao

4

6.4. Ket qua nang cao tiem luc khoa hoc

4

6.5. Danh gia tinh hinh thuc hien de tai nghien cuu

4

7. BAO CAO T O N G HOP

5

7.1. DAT VAN DE

5

7.2. TONG QUAN TAI LIEU NGHIEN ClTU

6

7.2.1. Benh viem gan B

6

7.2.2. Trieu chung lam sang cua benh viem gan virus B

6

7.2.3. Hau qua cua viem gan virus B

7

7.2.4. Suphan bo cac khu vuc nhilm HBV tren th^ giai

7

7.2.5. Virus gay benh viem gan B

9

7.2.5.1. Dac dien hinh thai cua virus HBV

9

7.2.5.2. CAu tao virus HBV

10

7.2.6. Cac chi thi cua virus viem gan B va cac xet nghiem chin doan hien nay . 12
7.2.6.1. Khang nguyen virus viem gan B va each phat hien

13

7.2.6.2. Phat hien cac khang th^ anti-HBV

13

7.2.7. Mot so phuang phap chan doan benh viem gan B

15

7.2.8. Hat latex va mot so ung dung

17

7.2.8.1 Giai thieu chung

17

7.3. MUC TIEU VA NO! DUNG NGHIEN ClTU

19

7.3.1. Muc tieu

19

7.3.2. Noi dung nghien cuu

^9

7.4. DIA DIEM, THOI GIAN VA PHUONG PHAP NGHIEN ClTU

20

7.4.1. Dia dilm, thai gian va phuang phap nghien cuu

20

7.4.1.1. Dia diem va thai gian

20

7.4.1.2. Vat lieu va phuang phap nghien cuu

20

7.4.1.2.1.Vat lieu

20

7.4.1.2.2. Phuang phap nghien cuu

21

7.4.1.2.2.1. Tach dong gen ma hoa cho khang nguyen HBc

21

7.4.1.2.2.2.Thiet ke vector bieu hien va bieu hien genHBc

21

7.4.1.2.2.3. Lam sach hat latex

22

7.4.1.2.2.4. Gan protein len be mat hat latex

22

7.4.1.2.2.5. Kiem tra tinh ngung ket ciia hat latex da gan protein len be mat

24

7.5. KET QUA NGHIEN ClTU

24

7.5.1. Phan lap gen ma hoa cho khang nguyen HBc

24

7.5.2. Thiet ke vector bieu hien va bieu hien gen HBc trong E.coli

25

7.5.3. Chuan hoa luong khang nguyen gan len be mat hat latex

27

7.5.4. So sanh kit ngung ket hat latex vai kit phat hien HBsAg

28

7.5.5. So sanh kit ngung ket hat latex vai kit phat hien HBcAg ciia Bio Rad

30

7.6. CAC CONG BO LIEN QUAN DEN KET QUA CUA DE TAI

31

7.7. KET QUA DAO TAO CUA DE TAI

31

7.8. KET QUA iTNG DUNG CUA D £ TAI

31

7.9. THAO LUAN

31

7.10. KET LUAN VA KIEN NGHI

32

7.10.1. Kit luan

32


7.1L TAI LIEU THAM KHAO

33

7.11.1. Tai lieu tilng Viet

33

7.11.2. Tai lieu tieng nuac ngoai

34

7.11.3.Cacwebside

35

8. PHU LUC

36

8.1. Qui trinh tach chiet ADN tong so tumau huyet thanh

37

8.2. So sanh trinh tu nucleotide ciia gen c

38

8.3. So sanh trinh tu amino acid ciia HBcAg

40

8.4. Ban sao cac cong bo lien quan den ket qua ciia de tai bai bao

41

8.5. Ban sao cac minh chiing huong dan khoa luan tot nghiep dai hoc

56

8.6. Ban sao de cuong va Hgp dong thuc hien de tai nghien ciiu
da dugc phe duyet

60

8.7. Tong quan ve de tai

72

8.8. Phieu tom tat ket qua nghien cuu cua de tai bang tieng Anh

75

8.9. Phieu dang ky ket qua nghien cuu KHCN

77


2. DANH MUC CAC TlT VIET TAT
RNA

Acid ribonucleic

DNA

Acid deoxyribonucleic

EDAC

l-Ethyl-3-(3-dimethylaminopropyl)
carbodiimide Hydrochloride

EIA

Enzyme imunoassay

ELISA

Enzyme linked immunosorbent assay

HBcAg

Hepatitis B core antigen

HBeAg

Hepatitis B e antigen

HBsAg

Hepatitis B surface antigen

HBV

Hepatitis B virus

Ig

Immunoglobulin

kDa

Kilo Dalton

RIA

Radioimmunoassay

PCR

Polymerase chain reaction


3. DANH SACH NGI/OfI THAM GIA THUC HIEN
- Chii tri dl tai:

TS. Chu Hoang Ha
Trung tam Cong nghe Sinh hpc phan tu
Truong dai hoc Cong nghe, Dai hoc Quoc gia Ha Noi
Vien Cong nghe Sinh hoc
Vien Khoa hpc va Cong nghe Viet Nam
Dien thoai: 7562368

- Nhijng nguoi tham gia thuc hien
PGS. TS. Le Tran Binh

Trung tam CNSHPT, Truong DHCN
Vien CNSH, Vien KH&CNVN
PGS.TS. Dinh Duy Khang Vien CNSH
TS. Le Van Son
Vien CNSH
CN. Phan Trpng Hoang
Vien CNSH
SV. Hoang Ha
Truong Dai hpc khoa hpc tu nhien

Dai hpc Quoc gia Ha Noi

4. DANH MUC CAC BANG SO LIEU
STT
01

Ten bang
Ket qua chan doan HBV bang kit ciia Bio Rad va bang hat latex gan
HBcAg

Trang
26

5. DANH MUC CAC HINH
STT

Ten hinh

Trang

01

Ban do ve sir phan bo cac khu vuc bi nhiem HBV a cac miic dp
khac nhau

8

02

Hinh dang ngoai ciia virus HBV

9

03

Vi tri ciia cac protein dupe ma hoa boi gen S

10

04

Genome cua HBV

11

05

Hat latex

17

06

Phan ling ngung ket giiia hat latex dupe gan protein voi khang the

18

07

Phan ling ngung ket giiia hat latex dupe gan khang the voi protein

18

08

Hat latex co nhom chiic dupe kich hoat

22

09

Gan protein len nhom chiic tren be mat hat latex

23
1


10

Quy trinh tien hanh ngung ket hat latex

24

11

PCR de nhan gen c

25

12

Cat cac dong vectbr tai to hop bang Sail va Hindlll

26

13

Protein HBcAg tai to hop sau khi tinh sach
Phan ling ngung ket giiia khang huyet thanh tho voi hat latex dupe
gan HBcAg

26

14

27

15

Thii ngung ket giua huyet thanh duong tinh voi kit thii phat hien
HBsAg voi hat latex gan HBc

28

16

Thai gian va miic dp xuat hien cac chi thi ciia virus viem gan B

29

17

Hien tuong ngung ket giixa huyet thanh am tinh voi kit thii phat hien
HBsAg vai hat latex gan HBc

29


6. TOM TAT CAC KET QUA NGHIEN ClTU CHINH CUA DE TAI
6.1. Ket qua khoa hoc
- Gen ma h6a cho khang nguyen tai tl hop HBcAg da dupe khuyech dai
thanh cong b4ng ky thuat PCR, dupe tach dong trong vector pBT, dpc trinh tu va
dang ky trinh tu gen gan hang dii lieu gen quoc te EMBL/GENBANK/DDBJ, ma so
AM888201.
- Gen c dupe gan thanh cong vao vector bieu hien pET21a(-H). Day la vector
CO gin them gen Trx tao dieu kien thuan Ipi cho viec bieu hien khang nguyen
HBcAg trong vi khuan E. coli va co duoi His-Tag thuan ipi cho viec tinh sach
protein tai to hop.
- Da nghien ciiu toi uu h6a quy trinh de gan protein tai to hpp HBcAg len be
mat hat latex. Hat latex dupe nia sach bang nuoc va buffer truoc khi'gan protein len
be mat. Nong dp HBcAg toi uu de gan len be mat hat latex la 400 |ig. Cac hat latex
dupe gan voi HBcAg cho ket qua ngung ket rat ro rang voi khang the khang
HBcAg CO trong huyet thanh ciia tho.
- Sii dung hat latex da gan HBcAg len be mat de ling dung trong chan doan
benh HBV. Kit ngung ket hat latex co dp nhay. dac hieu, va dp chinh xac cao khi so
sanh voi cac phuong phap thu nhanh (phat hien HBsAg) va ELISA (phat hien
khang the khang HBcAg). Kit ngung ket rat nhay trong trucmg hpp phat hien cac
benh nhan nhiem virus HBV 6 giai doan man tinh voi thao tac thuc hien don gian,
kinh te va tiet kiem thoi gian.
- Cong bo 01 bai bao dang tren tap chi Cong nghe Sinh hpc vai tieu de:

"Nghien cdu tao hat latex gan HBcAg de phdt hien /chdng the khdng HBcAg
trong huyet thanh benh nhan viem gan B'\ Cong trinh dang dupe ban bien tap
chinh siia va in an.
6.2. Ket qua phuc vu thirc te
Da sii dung hat latex gan khang nguyen HBc len be mat de chan doan thanh
cong benh HBV. Kit ngung ket hat latex co dp nhay, dac hieu, dp chinh xac cao,
thao tac don gian va rat nhanh. Co the ling dung bp kit nay de phat hien benh HBV
6 trong cac benh vien.


6.3. Ket qua dao tao
- Trong khuon kho de tai, 01 sinh vien da bao ve thanh cong khoa luan tot nghiep
06/2007 voi ten de tai: ^'Nghien ciru ky thuat gan khdng nguyen len be mat hat
latex kich thu&c nano vd irng dung trong viec chan dodn nhanh benh viem gan B
a ngu-&i'\
6.4. Ket qua nang cao tiem lye khoa hoc
- Tao dieu kien cho dpi ngii can bp tre tiep can, hpc hoi huong nghien ciiu trong
cong tac chan doan benh bang ky thuat ngung ket hat latex.
6.5. Danh gia tinh hinh thyc hien de tai nghien cihi
- De tai da hoan thanh tot cac noi dung nghien ciiu cung nhu dao tao theo hpp dong
so 59/HD-NCKH, ngay 5 thang 6 nam 2006. De tai da mo ra mot huong nghien ciiu
moi ling dung hat latex de phat trien cac kit chan doan benh.


7. BAO CAO TONG HOP
7,1. DAT VAN DE
Viem gan do HBV (hepatitis B virus) la mot benh truyen nhiem co mat o
moi viing dia ly va dang la moi quan tam ciia y te toan cau. Tren the gioi, co hon 2
ty nguoi nhiem HBV, hon 350 trieu nguoi mang HBV man tinh, trong do co 60
trieu nguoi chet vi ung thu gan va 45 trieu nguoi chet vi xa gan (To chiic y te the

gioi nam 1997) [26]. Viet Nam co so nguoi nhiem HBV cao voi ty le nhiem khang
nguyen bl mat HBsAg trung binh tii 10% - 20%. Neu tinh chung ca nhung nguoi
mang it nhat 1 trong 3 chi thi HBsAg, anti-HBs va anti-HBc thi ty le nhiem HBV
len toi 45% din 70%. Ty le viem gan c^p tinh do HBV hien chilm 37% - 49%, ty le
ung thu gan co HBV la 57 - 80 %. HBV dupe coi la tac nhan hang cjau gay ung thu
gan nguyen phat 6 nguoi [12].
De ngan chan su lay lan ciia benh va co cac bien phap dieu tri kip thai thi
viec chan doan nhanh va chinh xac viem gan B la rat can thiet. Cac phuong phap
chan doan viem gan B chu yeu la phat hien khang nguyen be mat ciia virus viem
gan B (HBsAg) va phat hien vat lieu di truyen (ADN) ciia virus. Tuy nhien, do hai
chi thi HBsAg va ADN khong phai luc nao cung ton tai trong huyet thanh nen rat
anh huong toi ket qua chan doan. Trong khi do, khang the khang HBcAg xuat hien
tu rat sdm va ton tai kha on dinh gan nhu suot cupc doi nguai benh. Day ro rang la
dau hieu chiing to sir khac biet giua nhCmg nguai da timg nhiem HBV voi nhung
nguai chua he bi nhiem. Lpi dung su co mat ciia khang the khang HBcAg de chan
doan HBV la hoan toan co co so va cho ket qua dang tin cay. Ket hpp vai cac
phuong phap da va dang sii dung de phat hien HBsAg se tao ra mot ngan hang mau
sach lam giam thieu nguy co nhiem HBV do truyen mau [19],
De nang cao hieu qua, tiet kiem thoi gian va tung buoc chii dpng dupe kit
chan doan, tii do co nhiing bien phap thich hpp nham som phong ngua benh, trong
de tai: 'Nghien cuu ky thuat gan khdng nguyen/khdng the len be mat hat latex
kich thw&c nano vd ung dung trong chan dodn nhanh benh if nguai va gia sue''
chiing toi chpn doi tupng benh viem gan B de tien hanh nghien ciiu. donu thai ma


ra mot huong nghien ciiu moi do la ling dung vat lieu nano trong nghien ciiu sinh
hpc tai Viet Nam.
7.2. TONG QUAN TAI LIEU NGHIEN ClTU
7.2.1. Benh viem gan B
Virus la mot trong nhung tac nhan chinh gay ra benh viem gan. Cac virus

nay chi tin cong vao gan, dan den ton thuong gan va huy hoai te bao gan. Cho den
cuoi thap ky 60 cua the ky XX, nguoi ta moi chi biet den 2 loai virus viem gan do la
viem gan dich tl (hay viem gan virus A) va viem gan huyet thanh (hay viem gan
virus B). Cho den nay, co 5 loai viem gan da dupe phat hien: viem gan do virus A,
B, D, E va G. Qua cac thong ke va cac nghien ciiu trong nuac cho thay d Viet Nam
da phat hien day dii cac loai virus viem gan. Trong do, viem gan do virus HBV la
pho bien hon ca.
7.2.2. Trieu chirng lam sang cua benh viem gan virus B
Khi benh nhan bj nhiem virus viem gan B thudng co hai trudng hpp xay ra:
Dgng cdp tinh: Dang cap tinh dupe bieu hien qua cac trieu chiing lam sang
nhu met moi, chan an, buon non, roi loan tieu hoa, sot nhe (38 C -38.5 C). Vai tuan
sau, thay nude tieu sam mau, phan c6 mau dat set, da vang. Mot so trudng hop benh
se tien trien sang the toi cap, hon me va chet sau vai ngay ke tir khi xuat hien cac
trieu chirng lam sang. Trudc khi xuat hien cac trieu chimg lam sang ke tren, ngudi
benh da phai trai qua thdi gian ii benh tir 2 tuan den 6 thang ma khong he biet minh
bi mac benh. Tai thdi ky nay, benh co the truyen tir ngudi nay sang ngudi khac ma
khong he hay biet [13].
Dgng man tinh: Dang man tinh khong bieu hien trieu chimg trong nhieu
nam, do vay benh ciing de bi lay cho ngudi than trong gia dinh va nhiJTig ngudi
Cling lam viec. Neu d the nhe, gan se bi thuong ton it, cac chirc nang ciia gan se
khong bi anh hudng. Neu d the nhiem dai dang, gan se bi ton thuong vira phai va
neu d the hoat dpng, gan se bi ton thuong sau sac, c6 the gay xo gan va sau do la
ung thu nguyen phat. Nhimg ngudi co benh d dang viem gan man tinh c6 t\ le tir
vong rat cao [13].


7.2.3. Hau qua cua viem gan virus B
Nhung ngudi bi viem gan man tinh se co nguy co chuyen thanh xo gan va
ung thu gan. Ty le tir vong ciia nhom ngudi nay la rat cao. Dac biet vdi cac tre nho,
neu nhiem virus viem gan B thi nguy co chuyen thanh viem gan man tinh rat Idn.

Hien nay, ty le tre em mang cac chi thj bieu hien nhiem HBV dang la moi lo ngai
sau sac doi vdi nhung quoc gia c6 ty le nhiem virus viem gan B cao, trong do co
Viet Nam. Vi vay, viec phat hien sdm va chinh xac benh de co nhirng bien phap
phong ngira thich hpp, nham giam nguy co lay lan cho cong dong, nhat la doi vdi
tre em, dang la van de dupe dat ra hang dau trong cac nghien cim khoa hpc.
7.2.4. Sir phan bo cac khu vuc nhiem HBV tren the gioi
Hien nay, su phan bo cac viing nhiem virus viem gan B dupe chia thanh 3
loai dua vao ty le nhiem virus viem gan B ciia tiing khu vuc tren the gidi.
Viing CO ty le nhiem cao: Qua cac thong ke cho thay co nhieu hem 8%o ngudi
bj nhiem HBV man tinh (tren ban do phan bo dupe bieu hien dudi dang mau do,
hinh 1). Nhung viing nay phan Idn d cac nude chau A (trir Nhat Ban va an Dp),
trong do co Viet Nam, chau Phi, hau het viing Trung Dong, viing chau tho Amazon
d Nam My, hau het cac nhom d quan dao Thai Binh Duong, tho dan chau Oc va dan
Maori [1].
Vidng CO ty le nhiem trung binh: Tai viing nay, ty le nhimg ngudi nhiem HBV
man tinh dao dpng tii 2% - S% (the hien bang mau xanh tren ban dl phan bl, hinh
1), gom CO cac nude nhu An Dp, mot phan Trung Dong, mien Tay A, Nhat Ban,
Dong Nam chau Au, va hau het mien Trung va Nam My.
Viing CO ty le nhiem thap: ty le nhung ngudi mang HBV man tinh luon nho
hon 2% (the hien bang mau xanh luc tren ban do phan bo, hinh 1). Nhimg vimg nay
bao gom: My, Canada, Uc, New Zealand va Tay Au.
Viet Nam nam trong khu vuc co ty le nhiem cao. Theo bao cao ciia Bp Y te,
tir cac nam 1978 den nam 1990 c6 trung binh khoang 20.000 ngudi/nam bj nhiem
HBV va ty le tir vong dao dpng tir 0,7% din 0,8%) [13],[12].
Theo cac nghien cim ciia GS. Pham Song, GS. Dao Dinh Diic va cong su,
nguyen nhan gay viem gan cap tinh trong nhimg benh nhan n^m dilu tri tai Vien Y
hpc Lam sang Nhiet ddi la do viem gan virus A. B, C, D va E, trong do co 26,31%
la do virus viem gan B [6].

>8% = savv. NHiCMCiO

Hinh 1. Ban do ve sy phan bo cac khu vtjc bi nhiem HBV a cac muc do khac nhau [ 1 ]

Theo ket qua nghien ciru ciia tac gia Le Thi Kim Dung va cong su cho
thay ty le ngudi mang HBsAg nam 2001 d Thanh phd Ho Chi Minh la 10%, d
Ha Noi la 14% [5].
Bang ky thuat ELISA, GS. Pham Song, GS. Do Trung Phan va cong sir dieu
tra tren 1865 ngudi me khoe manh trudc khi de thay 237/1865 ngudi (chiem 12,7%)
CO mang khang nguyen HBsAg, trong so nay thay 57 trudng hpp (chiem 36,3%)) co
ca HBeAg(+). Xet nghiem mau ciia tre so sinh d 237 ngudi me co HBsAg(+) cho
thay CO 106 tre so sinh mang ca hai loai khang nguyen HBsAg va HBeAg [3].
Ngay nay, moi lien he giira viem gan virus B phat trien thanh viem gan man
tinh, xo gan va ung thu gan nguyen phat da ro. Theo khao sat ciia tac gia Tran Thi
Lpi tai benh vien Tii Du - Thanh pho Ho Chi Minh, d benh nhi so sinh co tdi 85%
chuyen thanh viem gan man tinh, con ngudi Idn nguy co chuyen thanh viem gan
man tinh dao dpng trong khoang 5% - \0% [11].
Nhu vay cd the thay, d Viet Nam viem gan do virus viem gan B la can
nguyen chinh trong cac trudng hpp viem gan man tinh noi rieng va viem gan noi
chung.


7.2.5. Virus gay benh viem gan B
Hepatitis B virus (HBV) la mot thanh vien trong hp Hepadnaviridae gay
benh viem gan cho ngudi. Hien nay, hp Hepadnaviridae dupe xac dinh la hp virus
dpng vat, genome la ADN, co v6 ngoai va co the gay benh cho ngudi, dpng vat \a
chim. Cac virus trong hp nay co cau true va each sap xep genome rat giong nhau
nhung chi khac d moi quan he vat chii. Mac dii, cac virus hepadna deu co tinh
hudng gan nhung ADN ciia HBV cung dupe phat hien trong cac te bao tuyen tuy,
lach va than [10].

7.2.5.L Bac diem hinh thdi cua virus HBV
Nam 1964, Baruch Blumberg da mo ta mot loai khang nguyen dac trung ciia
tho dan chau Uc gpi la "khang nguyen Australia". Den nam 1968, phat hien trong
mau ciia benh nhan viem gan B man tinh co cac hat hinh cau va hinh soi co dudng
kinh 27 nm. Cac hat nay khong phai la HBV hoan chinh vi thieu he gen. Den nam
1970, ngudi ta phat hien trong mau benh nhan viem gan B c6 cac the hinh cau
dudng kinh len den 42 nm va ben trong co chira ADN kep. The hinh cau nay dupe
gpi la hat Dane (dat theo ten ngudi tim ra).

Hinh 2. Hinh dang ngoai cua virus HBV

Ghi chu: (I): Hat Dane hoan chinh; (2), (3): Phan bao ngoai dupe san xuit du thira
Qua cac nghien cim sau nay, cac nha khoa hpc da xac dinh dupe r^ng, hat
Dane mdi chinh la HBV thuc su. Cac hat hinh cau va hinh soi gin xung quanh la
khang nguyen be mat HBsAg. Ngoai ra, trong xet nghiem mau ciia benh nhan con
thay xuat hien cac hat hinh cau va hinh soi khong nam lien kit vdi hat Dane ma
nam le loi hoac dinh vao nhau thanh cum, do la thanh phan bao ngoai dupe san xu4l
du thira trong qua trinh nhan len ciia virus [10],[ 20].


7.2,5.2. Cdu tao virus HBV
cku tnic ciia HBV gom 3 lop: lop bao ngoai, Idp vd capxit va he gen
- Lap bao ngoai: dupe cau tao bdi 3 loai protein: Protein S (small), M
(middle), L (large)
L^^^Vi-'r?''''h^i\

--•-p'-ot*"'" "ho(S)
•p-ifc--— prote'ua kich ihifoc (ru^g binh
**
(S-t-PreSZ)

A ' l " protein c6 (cich thiTOclon
~"
(S +preS 1 • PreS2|

^*V

Hepatitis 8
virus

Hinh 3. Vi tri cua cac protein dugc ma hoa boi gen S [34]
Protein S (small) la protein nho nhat (trpng lupng phan tir la 24 kDa) trong
so cac protein be mat ciia virus, chiia 226 axit amin. Day la protein chinh hay con
gpi la SHBsAg dupe ma hoa bdi gen S va dupe ma hoa vdi so lupng Idn. Cac typ
ciia virus dupe xac dinh dua vao cac typ ciia cac SHBsAg nhd khang the don dong.
Protein M (middle) co kich thudc trung binh (281 axit amin), trpng lupng
phan tir la 31 kDa, do gen tien S2 va gen S ma hoa, hay con gpi la MHBsAg.
Protein L (large) la protein Idn nhat (39 kDa) chira 390 axit amin do cac gen
tien Si, tien S2 va S ma hoa, con dupe gpi la LHBsAg. Protein nay tham gia vao
viec gan virus vao vat chii, lap rap va giai phong virus khoi te bao [10], [16].
Vd Capxit:

Dupe cau thanh bdi hai loai chuoi polypeptide do la

polypeptide loi va tien loi.
Chuoi polypeptide loi co kich thudc ngan va trpng luong phan tir la 21 kDa.
Day chinh la khang nguyen loi ciia virus, ky' hieu la HBcAg (Hepatitis B core
Antigen).
Chuoi polypeptide tien loi co kich thudc dai han va trpng lupng la 25 kDa,
CO cau true bao gom toan bp chuoi ngan cong them mot peptide don co 29 axit

10


amin, duoc gpi la khang nguyen e ciia HBV (Hepatitis B e Antigen-HBeAg), day
chinh la san pham chuyen hoa ciia lap v6 capxit [2].
- He gen cua virus
Genome ciia HBV la phan tir DNA mach vong, chieu dai 3200 cap bazo va
CO cau triic mach kep khong hoan toan. Mach dai la mach ma hoa, con gpi la mach
am, day dii cac dau 5' - 3' xac dinh vdi mot phan du tan ciing gom 9 bazo, mach co
chieu dai co djnh la 3182 nucleotide. Cac loai mRNA va RNA tien gen dupe ma
hoa tir mach nay. Mach ngan hon con gpi la mach duong. Chieu dai ciia mach nay
chi bang 50-80%) mach am. Dau tan cimg 5' ciia mach duong dupe tao bdi mot
oligoribonucleotide chieu dai 18 bazo va duoc gan mu tuong tu nhu mRNA [20],
[25].
Gen S (surface): Ma hoa cho khang nguyen be mat HBsAg, bao gom 3
phan: Tien SI hay con dupe gpi la preSl, tien S2 hay con gpi la preS2 va S. Vimg S
va tien S2 co chieu dai co dinh con viing SI co chieu dai thay doi tuy theo typ virus.
Cac polypeptide be mat ciia HBV co lien quan vdi nhau vi deu co chung viing S.
Cac gen S ma hoa cho 3 loai protein khac nhau do la S, M va L.

PfBS2

PnC

:-'r. o^-,-'w.'.';.-J o-.irf.'

Hinh 4. Genome cua HBV [ 1 ]
He gen ciia HBV chira cac gen S, C, P va X. Trong do:

II

Gen C (core): la viing ma hoa cho protein nucleocapsit. Gen C co hai viing
d6 la viing loi C va viing tien loi dupe gpi la preC. Hai vimg nay ma hoa cho hai
loai protein la protein loi va protein tien loi [25].
Protein loi la chuoi ngan (21 kDa) gom 138 axit amin, co kha nang gan vdi
axit nucleic. Day la khang nguyen loi HBcAg. Do khong co doan peptit tin hieu nen
khong tiet ra khoi te bao gan nhung tham gia vao qua trinh lap rap virus.
Protein tien loi la mot polypeptide dai 25 kDa gom 212 axit amin. Protein
nay dupe cau tao bdi toan bp chuoi ngan duoc cong them 29 axit amin do viing tien
loi ma hoa. Protein tien loi c6 doan peptide tin hieu nam d dau amin. Chinh doan do
se hudng chuoi polypeptide tien loi mdi tong hpp vao mang ludi noi chat ciia te
bao. Tai day, chiing dupe xu ly bang each loai bo 19 axit amin d dau amin khoang
36 axit amin d phia dau carboxyl, sau do dupe tiet ra ngoai te bao dudi dang
HBeAg [10], [1].
Gen P: la gen Idn nhat, chiem 80% chieu dai ciia genom, ma hoa cho chuoi
polypeptide Idn co trpng lupng len den 90kDa gom 832 axit amin rat giau histidine.
Protein nay co hai hoat tinh: ADN polymerase va enzym phien ma ngupc (DNA
polymerase phu thuoc vao RNA).
Protein P c6 hai viing chirc nang: Viing thir nhat la viing protein cuoi nam d
dau amin va viing thir hai co hoat tinh ribonuclease H nam d dau carboxyl. Protein
P cung tham gia vao thanh phan cau tao nen cau triic virus [ I], [10].
Gen X: la gen co khung dpc md nho nhat. Gen nay ma hoa cho protein X hay
HBx. Day la mot protein chira 154 axit amin. Protein X c6 nhieu chirc nang khac
nhau nhu tuong tac vdi bp may phien ma, tham gia vao viec truyen tin hieu, chuyin
dang te bao gan thanh te bao ung thu... Protein HBx co the gan true tiep vao p53 dl
ngan can chirc nang ciia protein nay va ngan can qua trinh sira chiia ADN sai hong
theo CO che cat bo nucleotit [1], [22].
7.2.6. Cac chi thi cua virus viem gan B va cac xet nghiem chan doan hien nay
Khi nhiem benh, trong huyet thanh ciia benh nhan mot vai tuan sau khi nhiem

se xuat hien cac chi thi (marker) bieu hien qua trinh phat trien ciia virus trong co thi
ciing nhu su phat trien ciia benh. Qua cac nghien cim thong ke, cac nha nghien cim da
xac dinh dupe thdi gian phat hien cung nhu bien mat cua cac chi thi (marker) trong ca
the ngudi benh. Tii dd, co the du doan tinh trang ciia benh nhan sau khi nhiem HBV.

12


7.2,6,L Khdng nguyen virus viem gan B vd cdch phdt hien
- HBsAg la khang nguyen bl mat ciia HBV, xuat hien rat sdm trudc khi co
cac trieu chimg lam sang, nong dp tang cao dan va bien mat sau 4 den 8 tuan ke tir
khi CO trieu chimg. N I U sau 6 thang ma van con HBsAg thi co nguy co chuyen
thanh ngudi mang HBsAg man tinh. Sir hien dien ciia HBsAg trong huyet thanh
chiing to su co mat ciia ADN ciia virus trong te bao gan. Neu trong giai doan binh
phuc ham lupng HBsAg giam hin so vdi tri sd ban dau thi co nguy co trd thanh
ngudi mang HBV man tinh [4].
De phat hien khang nguyen HBsAg, hien nay co nhieu loai xet nghiem cho
ket

chinh

xac

nhu

(Radioimmunoassay),

ELISA

(Enzyme

linked

immunosorbent

assay),

RIA

EIA (Enzyme imunoassay)...Nhung day la nhung phuang

phap chan doan trong phong thi nghiem doi hoi ton nhieu thdi gian nhung ket qua
rat chinh xac. Hien nay, cac cong ty tren the gidi da phat trien he thong cac kit dudi
dang que thir de chan doan nhanh benh HBV nhu Cyprus Diagnostics [29], Akers
Laboratories [30]...
+ Khang nguyen HBeAg xuat hien muon d giai doan binh phuc. No la dau hieu
ciia su khoi benh. Cac phuong phap phat hien thay khang nguyen nay gom ky thuat
danh dau phong xa, ky thuat huynh quang mien dich [1], [4].
- Khang nguyen HBcAg chi xuat hien trong te bao gan va chi dupe phat hien
khi lam sinh thiet gan. HBcAg khong xuat hien trong huyet thanh vi v6 ngoai co
HBsAg bao quanh lay no va HBcAg thieu mot doan tin hieu lam cho HBsAg khong
the duoc chuyen ra ngoai ludi noi chat de ra khoi te bao [4].
7.2.6.2. Phdt hien cdc khdng the anti-HBV
Co kha nhieu cac khang the khang HBV co the phat hien dupe trong huyet
thanh va huyet tuong bang cac each phat hien khac nhau.
+ Khang the anti-HBs xuat hien muon tir 2 den 16 tuan sau khi khong phat
hien thay HBsAg. Anti-HBs IgM xuat hien trong giai doan cap, con anti-HBs IgG
xuat hien muon hon nhung ton tai lau hon. Su xuat hien anli- HBs la dau hieu benh
da dupe cai thien va nd co tac dung chong tai nhiem HBV.

13


Khang the anti-HBs thudng ton tai suot cupc ddi ngudi benh, nhung chiing co
the khong dupe phat hien sau mot so nam.
Cac khang the khang HBsAg dupe phat hien trong phdng thi nghiem bang
cac phuang phap nhu RIA, ELISA... Trong d6, HBsAg dupe co dinh vao mot pha
ran va dupe danh dau phong xa bdi '^^I hoac enzym. Hai dau hoat dpng ciia khang the
tao nen cau ndi giua khang nguyen pha ran vdi khang nguyen dupe danh dau [4].
+ Khang the tdng so khang HBc la nhiing khang the khang true tiep vdi cac
epitope protein vd ciia virus HBV. Chung xuat hien sdm trong giai doan bi nhiem
benh (khoang 2 den 3 tuan sau khi nhiem benh) va ton tai lau dai trong suot cupc
ddi ngudi benh ngay ca khi khong co mat ciia HBsAg va khang the anti-HBs. Cac
khang the nay dupe phat hien rat on dinh do do chiing rat co lpi khi dupe img dung
trong chan doan. Nhung su xuat hien ciia khang the anti-HBc khong co tac dung
ching tai nhiem HBV[l], [32].
Trong thdi ky dau thong thudng xuat hien khang the anti-HBc IgM vdi nong
dp cao. Nhirng ngudi bi nhiem virus viem gan the cap tinh thudng co nong dp antiHBc IgM cao hon nhieu lan nong dp anti-HBc IgG. Con vdi nhiing benh nhan bi viem
gan do virus viem gan B d dang man tinh, thong thudng ndng dp anti-HBc IgG cao
hon vi khang the anti- HBc IgG luon xuat hien sau khang the anti- HBc IgM khoang 3 4 tuan. Cac xet nghiem anti-HBc IgM dupe diing trong chan doan benh cap tinh.
Hien nay, c6 rat nhieu phuang phap diing de phat hien khang the anti-HBc nhu ky
thuat dien di mien dich, ky thuat mien dich huynh quang, ky thuat ngung kit... [1],
[18].
Doi vdi cac benh nhan bi nhiem bdi cac chiing HBV hoang dai thi khang thi
anti-HBe xuat hien sau khi HBeAg bien mat. Trong thdi ky nay thi su sao chep ciia
virus da ngirng do chu ky tu nhien hoac do dieu tri khang virus thanh cong. D6i vdi
cac trudng hpp bi nhiem phai chiing virus dot bien tien nhan thi khang thi anti-HBe
xuat hien ca khi virus dang sao chep va khong con sao chep. Vi vay, viec phat hien
ADN ciia HBV la can thiet de phan biet cac benh rihan bi nhiem chiing virus dang
gi [32]. Thdng thudng khang the anti-HBe IgM xuat hien sdm con khang thi anti-HBe

IgG xuat hien muon hon [1], [4].

14


7,2.7. Mot sd phuang phdp chan dodn benh viem gan B
Sir tiln bp trong cac linh vuc sinh hpc phan tir, mien dich hpc, virus hpc... da
hinh thanh nhilu nghien cim giiip chan doan nhanh va chinh xac benh viem gan B.
Dudi day la mot s6 ky thuat trong ITnh vuc sinh hpc phan tir va mien dich hpc nham
phat hien nhanh benh viem gan B.
+ Phuangphdp

dien di mien dich doi luu (Counter

Immunoelectrophoresis-CIEP)

Khang nguyen tich dien am tu gieng d phia cue am, khang the tich dien
duong tir gieng d phia cue duong, chiing di chuyen ngupc nhau, khi gap nhau se
hinh thanh kit tiia [1], [9].
+ Phuang phdp tham mien dich (Western Blot)
Ky thuat nay dupe diing nhieu trong chan doan nhiem cac benh do virus,
nhiem khuan, xac dinh khang the don dong. Ky thuat gom 3 budc:
Budc 1: Tien hanh dien di hon hop dung dich protein hoa tan tren gel
polyacrylamide de tach peptide hoac cac tieu phan protein khac nhd lire dien trudng
Budc 2: Dimg mang nitrocellulose tham len ban gel de chuyen cac chuoi
peptide ra khoi ban gel va di vao mang nitrocellulose
Budc 3: Phil hon hpp chira khang the dac hieu vdi khang nguyen len tren
mang nitrocellulose roi ii. Rira khang the thira khong tham gia phan ling. Them
khang the da gan enzym, them co chat tuong img va chat phan irng mau. Do cudng
dp mau de danh gia mire dp enzym phan giai co chat [9].

+ Phuang phdp tgo tiia (Precipitation test)
Khi cho khang the dac hieu phan img vdi khang nguyen hoa tan d lieu lupng
chuan se xuat hien ket tiia co the nhin thay bang mat thudng. Phan img nay dupe
dimg kha pho bien de phat hien khang nguyen khi da co san khang the dac hieu
hoac de phat hien khang the khi da c6 san khang nguyen hoa tan dac hieu. Phan img
ket tua bi ire che khi co qua nhieu khang nguyen hoac khang the. Ket tiia dat toi uu
khi nong dp khang nguyen - khang the thich hpp. Khi ty le tuong duong se tao
thanh mang ludi ket tiia tot nhat [9].
+ Phuang phdp dien di mien dich

(Immunoelectrophoresis)

15


Dudi tac dung ciia dong dien, cac thanh phan khang nguyen mang dien tich
se di dpng tren gel agarose, van tdc va khoang each phu thuoc vao thanh phan
khang nguyen. Sau khi cac thanh phan khang nguyen dupe phan tach bdi dong dien,
khang the dupe dat vao ranh chay song song vdi hudng dien di. Tii ranh nay khang
the dupe khuech tan ve phia khang nguyen va ngupc lai. Ket tiia la dac hieu khi co
ty le nong dp toi uu khang nguyen-khang the va hinh thanh cac vach ket tiia tren
ban gel [9].
+ Phuang phdp huynh quang mien dich (Immunofluorescent

technique)

Trong ky thuat nay khang the hoac khang nguyen dupe danh dau bang thuoc
nhupm huynh quang. Ky thuat dua vao tinh chat ciia thuoc nhupm khi bi kich thich
bdi hire xa cd budc song dac biet se phat sang. Mot so chat nhu rodamin phat huynh
quang mau dd da cam, fluorescein phat huynh quang mau vang luc [9]

+ Phuang phdp mien dich phong xg (Radioimmunoasay

~ RIA)

Day la ky thuat do Rosalyn Yalowd dua ra nam 1960 de xac dinh nong dp
insulin trong huyet tuong. Ngay nay, ky thuat nay duoc diing de xac dinh ndng dp
huyet tuong, thuoc, globulin mien dich, cac khang nguyen virus... Phuong phap
nay dua tren sir canh tranh giiia khang nguyen danh dau dong vi phong xa (vi du
I) vdi khang nguyen khong danh dau dong vi phdng xa dupe gan vao khang the
dac hieu [9].
+ Phuang phdp mien dich lien ket enzym (Enzyme linked immunosorbent

assay-

ELISA)
Nguyen tac ciia ky thuat nay giong nhu ky thuat mien dich huynh quang
nhung thay vi gan khang the len thuoc nhupm huynh quang thi ngudi ta gan khang
the vdi mot enzym. Khi co them mot co chat thich hpp vao phan img, enzym se
thuy phan co chat thanh mot chat co mau. Sir xuat hien mau chimg to da c6 phan
irng dac hieu giira khang the vdi khang nguyen va thong qua cudng dp mau co the
biet dupe nong dp khang the hay khang nguyen can xac dinh [9].
+ Phuang phdp ngung ket (Agglutination

test)

(j ky thuat nay can c6 khang nguyen huu hinh (particulate). Bo la khang
nguyen co kich thudc Idn nhu hong cau va te bao \'i sinh vat. Khang nguyen hiru
hinh CO cac epitop be mat c6 the lien ket cheo vdi khang the tao ra timg cum nhin
dupe bang mat thudng. Khang the trong phan irng nay gpi la khang the gay ngung

kit. Phan ling ngung kit nhay hon phan img ket tiia nen dupe diing de dinh tinh va
ban dinh luong khang thi trong huylt thanh [11], [12]. Phuong phap cai tien cua
phuang phap nay gpi la ngung ket gian tiep hay ngung ket thu dpng, co the gay
ngung ket khang nguyen hoa tan nhung trudc do da gan no len tren be mat cac chat
tra huu hinh nhu hong cau, hat bentonit hoac hat latex [15].
7.2.8. Hat latex va mot so irng dung
7.2.8.1. Gi&i thieu chung
Hat latex thudng dupe hinh thanh bang each triing hpp nhu tuong. Qua trinh
duoc bat dau bang viec trpn styrene vdi dung dich cac chat co hoat tinh be mat nhu
natri dodecyl sulfat sau do cho cac hat mixen vao hon hpp. Ket qua la sir nhu tuong
hoa hang trieu hat mixen dong nhat ve dudng kinh. Tiep do, cho them mot vai chat
khdi dau cho qua trinh triing hpp nhu chat kali persulfat. Khi qua trinh triing hpp da
hoan thanh, cac chuoi polystyrene dupe gan vao trong cac hat mixen va cac ion
sulfat phan duoi d tren be mat ciia khoi cau.
Cac hat latex c6 the duoc tao thanh vdi dudng kinh mong muon bang each
thay doi hydrocarbon, chat hoat dpng be mat, chat khdi dau va qua trinh chuan bj.
Ngoai polystyrene (PS) hat latex con dupe tao ra tir styrene vinyl toluene,
silica...va co kich thudc dao dpng tii 0,02-1000 |im [31].

Hinh 5. Hat latex [17]

Thdng thudng hat latex co hai loai: mot loai khong co nhom chirc tren bl mat
va mot loai co nhom chirc tren be mat nhu: amino (NH2), hydroxyl (OH), carboxyl
(COOH)... Tiiy theo muc dich irng dung khac nhau ma ngudi ta co the chpn loai hat
CO hoac khong chira nhom chiic. Hien nay, nhom hat latex co nhom chuc tren b^

DAI HOC OUOC GIA HA NOI
TRUNG TAM THONG TIN I H U VIEN

OO060O00O5"5'


mat duoc sir dung rong rai hon ca vi chiing cho phep cac thanh phan gan len no ben
vurng va ch^c chin. Cac loai hat latex c6 nh6m chirc khac nhau thi qua trinh gan
protein len bl mat ciia chiing ciing khac nhau. Doi vdi loai hat chira nhom OH thi
c4n cyanogen bromide lam xiic tac hoac neu la nhom chirc COOH thi phai can chat
EDAC. Ben canh d6 cac hat latex cung da dang vl mau sac de de dang dpc ket qua
bang mat thudng [31].
7.2.8.2. Mot s6 dng dung cda hat latex
Cac hat latex co kich thudc tir 0,05^m-2^m, be mat dupe gan vdi cac phan tir
protein khang nguyen tai to hpp hoac cac phan tir khang the. Neu hat latex dupe gan
khang nguyen len bl mat thi se dupe irng dung de phat hien khang the co chiia trong
mau can xet nghiem. Nguyen tac dupe mieu ta d hinh 6.
Khang the

Hat latex gan HBc.Ag lien ket voi
khang the tao hien tuang ngung ket

Hat latex gan HBcAg 6 dang ty

Hinh 6. Phan ling ngung kel giiia hat latex dugc gan protein voi khang the
• : khang nguyen; J ^ : khang the dac hieu vai khang ngu>en •• ; Y

latex dugc gan vai khang nguyen

Neu hat latex dupe gan khang the len be mat thi chiing se dupe img dung de
phat hien cac khang nguyen co trong mau nghien cuu. Nguyen tac dupe the hien d
hinh 7.

Khane neuven
•

Hinh 7. Phan ung ngung ket giua hat latex dugc gan khang the vai protein
khang nguyen; J^: khang the dac hieu vai khang nguyen H H ^ p

18

latex dugc gan vai khang the


Nhu vay, ban chit la su ngung ket giira khang nguyen va khang the trong do
hat latex ddng vai tro la gia do lam cho cac lien ket nay trd nen on dinh hon. Liic
nay, phan img ngung kit thuc chit la mot phan img triing hpp ciia cac hat latex co
kich thudc nho. Miic dp ngung ket phu thuoc nhieu vao nong dp ciia khang the
trong mau nghien cim va so protein dupe gan tren be mat ciia hat latex [21], [23].
Xet nghiem ngung ket hat latex lan dau tien dupe Singer va Plotz (1956) img
dung thanh cong de chan doan viem khdp (Rheumatoid). Tir do den nay, hat latex
da dupe img dung rong rai trong chan doan mien dich va da dupe irng dung de phat
hien hon 100 loai benh nhiem triing [23]. Bang each gan khang nguyen len be mat
hat latex, Sukone va Janya (2005) [24]; Yamamoto va dong tac gia (2000) [28]; Xu
va dong tac gia (2005) [27] ciing da chan doan dupe benh sot vang da
(Leptospirosis), benh viem nao Nhat Ban va benh ciim gia cam (H5N1) ...
Hien nay, cac xet nghiem mdi co sir dung cac thiet bj do dac hien dai da lam
cho ky thuat ngung ket latex co dp chinh xac rat cao va trd thanh mot xet nghiem
mien dich dinh lupng tu dpng hoa. Do vay, cac xet nghiem sir dung hat latex van
dang tiep tuc nghien cim va phat trien nhu xet nghiem dai cromatographic hay xet
nghiem bat giii" tieu the mang [17], [21].
7.3. MUC TIEU VA N Q I DUNG NGHIEN ClTU
7.3.1. Muc tieu

Tao ra dupe hat latex gan khang nguyen tai to hpp HBcAg de ling diing
trong chan doan nhanh benh virus viem gan B (HBV)
7.3.2. Noi dung nghien ciru
+ Phan lap gen ma hoa cho khang nguyen HBc
ADN ciia virus gay benh viem gan B dupe tach chiet. Thiet ke cap moi dac
hieu de khuyech dai dupe gen ma hoa cho khang nguyen HBc ciia virus viem gan B
bang ky thuat PCR. Tii do tach dong va xac dinh trinh tu nucleotit ciia gen.
+ Thiet ke vector bieu hien
Gen ma hoa cho khang nguyen HBc dupe gan vao vector bieu hien pET
21a(-i-) dupe sir dung la vector bieu hien mang gen cau triic vi c6 77 promotor va

19


Trx lam tang cudng kha nang bieu hien protein dich va duoi His-Tag thuan tien cho
viec tinh sach protein tai to hop.
+ Tinh sach protein tai to hpp
Protein tai tl hpp dupe bieu hien dudi cac dieu kien toi uu se dupe tinh sach
theo phuang phap sac ky ai luc.
+ Xac dinh dupe nong dp khang nguyen HBcAg thich hpp nhat de gan hieu qua len
be mat hat latex.
+ iTng dung cac hat latex da dupe gin HBcAg de phat hien khang the khang
HBcAg c6 trong huyet thanh ciia cac benh nhan nhiem HBV.
7.4. DIA DIEM, THOTI GIAN VA PHlTONG PHAP NGHIEN ClTU
7.4.1. Dia diem, thoi gian va phuong phap nghien ciru
7.4.1.1. Dia diem va thoi gian
Cong trinh dupe thuc hien tai Trung tam Cong nghe Sinh hpc phan tir, DH
Cong nghe va tai phong Cong nghe Te bao Thuc vat, Vien Cong nghe Sinh hpc,
Vien Khoa hpc va Cong nghe Viet Nam tir 06/2006-06/2007.
7.4.1.2. Vat lieu va phirffng phap nghien ciru

7,4.l.2.l.Vgtlieu
Huyet thanh ciia ngudi do Vien Huyet hpc truyen mau Trung uong cung cap:
05 mau huyet thanh duong tinh vdi kit thir phat hien su co mat ciia HBsAg va 05
mau huyet thanh khong phat hien co HBsAg. 20 mau huyet thanh duang tinh vdi kit
phat hien su co mat anti-HBcAg ciia hang Bio Rad (My, thir bang phuong phap
ELISA) va 20 mau huyet thanh am tinh vdi kit nay. Huyet thanh ciia tho trudc, sau
gay mien dich vdi HBcAg; va khang nguyen HBc tai to hpp tinh sach sir dung trong
cong trinh nay duoc Nguyen Tien Minh va dong tac gia (2003) nghien ciru trudc tai
phong Vi sinh vat hpc phan tir vien Cong nghe sinh hpc.
Hoa chat: PolyLink Coupling Buffer (50ml), PolyLink wash/Storage buffer
(45ml), PolyLinhk EDAC (Carbodiimide) (750mg), hat latex kich thudc 1,1 [im do
hang Bangs Labratories, Inc cung cap. Vector pET 21a(+), chiing te bao bieu hien
Rosseta gami co 6 bd ba ma hiem dupe cung cap bdi Novagen. Marker protein,
ADN, IPTG dupe cung cap bdi Fermentas. Kit PCR, vector tach dong pBT do

20