SỞ KHOA HỌC VÀ CÔNG NGHỆ
THÀNH ĐOÀN TP.HCM
CHƯƠNG TRÌNH VƯỜN ƯƠM SÁNG TẠO KH-CN TRẺ
---------------------------------BÁO CÁO NGHIỆM THU
(ÑAÕ CHÆNH SÖÛA THEO GOÙP YÙ CUÛA HOÄI ÑOÀNG NGHIEÄM THU)
ÑEÀ TAØI
SÖÛ DUÏNG KYÕ THUAÄT NESTED PCR – RFLP ÑEÅ XAÙC ÑÒNH
ÑOÄT BIEÁN KHAÙNG LAMIVUDINE VAØ ÑOÄT BIEÁN BASAL
CORE PROMOTER CUÛA VIRUS VIEÂM GAN B
CHỦ NHIỆM ĐỀ TÀI: ThS. NGUYEÃN CHÍ VINH
CƠ QUAN CHỦ TRÌ: TT PHAÙT TRIEÅN KHOA HOÏC COÂNG NGHEÄ TREÛ
THÀNH PHỐ HỒ CHÍ MINH
THÁNG 01/ 2008
SỞ KHOA HỌC VÀ CÔNG NGHỆ
THÀNH ĐOÀN TP.HCM
CHƯƠNG TRÌNH VƯỜN ƯƠM SÁNG TẠO KH-CN TRẺ
---------------------------------BÁO CÁO NGHIỆM THU
(ÑAÕ CHÆNH SÖÛA THEO GOÙP YÙ CUÛA HOÄI ÑOÀNG NGHIEÄM THU)
ÑEÀ TAØI
SÖÛ DUÏNG KYÕ THUAÄT NESTED PCR – RFLP ÑEÅ XAÙC ÑÒNH
ÑOÄT BIEÁN KHAÙNG LAMIVUDINE VAØ ÑOÄT BIEÁN BASAL
CORE PROMOTER CUÛA VIRUS VIEÂM GAN B
CHỦ NHIỆM ĐỀ TÀI: ThS. NGUYEÃN CHÍ VINH
CƠ QUAN CHỦ TRÌ: TT PHAÙT TRIEÅN KHOA HOÏC COÂNG NGHEÄ TREÛ
COÁ VAÁN CHUYEÂN MOÂN:
1. PGS.TS HOÀ HUYØNH THUØY DÖÔNG
2. GS.BSCKII PHAÏM HOAØNG PHIEÄT
THÀNH PHỐ HỒ CHÍ MINH
THÁNG 01/ 2008
THÔØI GIAN THÖÏC HIEÄN ÑEÀ TAØI:
09/2006 – 01/2008
ÑÒA ÑIEÅM THÖÏC HIEÄN ÑEÀ TAØI:
Khoa Xeùt nghieäm - Beänh vieän Ñaïi hoïc Y Döôïc TP.HCM
Boä moân Di truyeàn - Ñaïi hoïc Khoa hoïc Töï nhieân TP.HCM
THAØNH PHAÀN THAM GIA:
ThS. Nguyeãn Chí Vinh: Khoa Xeùt nghieäm – Beänh vieän Ñaïi hoïc Y döôïc
CN. Hoaøng Ngoïc Baûo Mi: Khoa Xeùt nghieäm – Beänh vieän ÑH Y döôïc
CN. Döông Thò Thanh Höông: Khoa Xeùt nghieäm – Beänh vieän ÑH Y döôïc
CN. Hoà Thò Thanh Thuûy: Boä moân Di truyeàn – ÑH Khoa hoïc Töï nhieân
CN. Nguyeãn Vuõ Trung Kieân: Boä moân Di truyeàn – ÑH Khoa hoïc Töï nhieân
CN. Ngoâ Thò Thanh Tuyeàn: Trung taâm xeùt nghieäm Diag Center
COÁ VAÁN CHUYEÂN MOÂN:
PGS.TS Hoà Huyønh Thuøy Döông – Boä moân Di truyeàn
Ñaïi hoïc Khoa hoïc Töï nhieân TP.HCM
GS.BSCKII Phaïm Hoaøng Phieät – Khoa Xeùt nghieäm
Beänh vieän Ñaïi hoïc Y döôïc TP.HCM
Baûng ghi chuù caùc chöõ vieát taét:
Antiviral
BCP
BET
bp
DNA
dATP
dCTP
dGTP
dTTP
dUTP
dNTP
DW
EDTA
HBV
IFN
LAM
M
Mut
Nu
OLT
PCR
Phage
Plasmid
Primer
RFLP
rpm
Sequencing
Taq
V
YMDD
Wt
: Thuoác khaùng virus
: Basal core promoter
: Ethidium bromide
: base pair – caëp base
: Deoxyribonucleic acid
: 2’-deoxyadenosin- 5’- triphosphate
: 2’-deoxycytidin- 5’- triphosphate
: 2’-deoxyguanosin- 5’- triphosphate
: 2’-deoxythymidin- 5’- triphosphate
: 2’-deoxyuridin- 5’- triphosphate
: 2’-deoxynucleosid- 5’- triphosphate
: Distilled water – nöôùc caát voâ truøng
: Ethyldiamin tetra-acetic acid
: Hepatitis B Virus, Virus vieâm gan B
: Interferon
: Lamivudine
: Methionine
: Mutation – ñoät bieán
: Nucleotide
: Orthotopic liver transplant – gheùp gan
: Polymerase chain reaction
: Thöïc khuaån theå
: Theå mang DNA muïc tieâu – duøng trong kyõ thuaät taïo doøng
: Moài – duøng trong phaûn öùng PCR
: Restriction fragment length polymorphisms –
tính ña hình kích thöôùc cuûa caùc trình töï giôùi haïn
: rounds per minute – voøng treân phuùt
: Giaûi trình töï nucleotide
: Thermus aquaticus
: Valine
: Tyrosine Methionine Aspartate Aspartate
: Wild type – hoang daïi
PHAÀN 1:
TOÅNG QUAN TAØI LIEÄU
PHAÀN 2:
NOÄI DUNG – PHÖÔNG PHAÙP
PHAÀN 3:
KEÁT QUAÛ – BAØN LUAÄN
MUÏC LUÏC
Trang
Baûng ghi chuù caùc chöõ vieát taét
Muïc luïc
PHAÀN 1: TOÅNG QUAN TAØI LIEÄU
1. Vieät Nam thuoäc vuøng nhieãm vaø beänh lyù do HBV naëng, soá löôïng beänh
nhaân coù chæ ñònh ñieàu trò lôùn
2. Chaån ñoaùn ñöôïc ñoät bieán Basal core promoter vaø ñoät bieán khaùng
lamivudine seõ giuùp ích cho vieäc theo doõi vaø ñieàu trò beänh vieâm gan B
3. Nested PCR-RFLP laø kyõ thuaät ñôn giaûn vaø tieän ích ñeå xaùc ñònh ñoät bieán
Basal core promoter vaø ñoät bieán khaùng lamivudine
1
1
7
PHAÀN 2: NOÄI DUNG – PHÖÔNG PHAÙP
1. Taïo vaät lieäu cho quy trình
1.1. Taïo saûn phaåm Nested PCR chöùng döông cuûa ñoät bieán Basal core
promoter
1.2. Taïo doøng löu tröõ caùc daïng cô baûn cuûa ñoät bieán khaùng lamivudine,
töø ñoù taïo caùc saûn phaåm PCR chöùng döông
2. Thöû nghieäm thay ñoåi moät soá yeáu toá
2.1. Taùch chieát DNA baèng phöông phaùp tuûa thay cho phöông phaùp
Boom
2.2. Nested PCR thay cho PCR 1 böôùc
2.3. Phaûn öùng uû thích hôïp cho caùc enzyme giôùi haïn
2.4. Ñieän di microfluidic thay cho ñieän di agarose 3%
3. Thöû khaû naêng phaùt hieän ñoät bieán trong huyeát thanh coù hoãn hôïp HBV ñoät
bieán/hoang daïi
3.1. Khaû naêng phaùt hieän ñoät bieán Basal core promoter trong huyeát thanh
hoãn hôïp
3.2. Khaû naêng phaùt hieän ñoät bieán khaùng lamivudine trong huyeát thanh
hoãn hôïp
3.3. Ñoä nhaïy cuûa quy trình xaùc ñònh ñoät bieán khaùng lamivudine
4. Thöïc hieän 2 quy trình ñaõ ñöôïc caûi tieán treân huyeát thanh beänh nhaân
4.1. Xaùc ñònh ñoät bieán Basal core promoter treân huyeát thanh beänh nhaân
4.2. Xaùc ñònh ñoät bieán khaùng lamivudine treân huyeát thanh beänh nhaân
14
14
PHAÀN 3: KEÁT QUAÛ – THAÛO LUAÄN
1. Keát quaû veà vieäc taïo vaät lieäu cho quy trình
1.1. Taïo saûn phaåm Nested PCR chöùng döông cuûa ñoät bieán Basal core
15
8
8
9
10
11
12
12
13
13
promoter
1.2. Taïo doøng löu tröõ caùc daïng cô baûn cuûa ñoät bieán khaùng lamivudine,
töø ñoù taïo caùc saûn phaåm PCR chöùng döông
2. Keát quaû veà vieäc thöû nghieäm thay ñoåi moät soá yeáu toá
2.1. Taùch chieát DNA baèng phöông phaùp tuûa thay cho phöông phaùp
Boom
2.2. Nested PCR thay cho PCR 1 böôùc
2.3. Phaûn öùng uû thích hôïp cho caùc enzyme giôùi haïn
2.4. Ñieän di microfluidic thay cho ñieän di agarose 3%
3. Keát quaû veà vieäc thöû khaû naêng phaùt hieän ñoät bieán trong huyeát thanh coù
hoãn hôïp HBV ñoät bieán/hoang daïi
3.1. Khaû naêng phaùt hieän ñoät bieán trong huyeát thanh hoãn hôïp
3.1.1 Khaû naêng phaùt hieän ñoät bieán Basal core promoter trong huyeát
thanh hoãn hôïp
3.1.2. Khaû naêng phaùt hieän ñoät bieán khaùng lamivudine trong huyeát
thanh hoãn hôïp
3.2. Ñoä nhaïy cuûa quy trình xaùc ñònh ñoät bieán khaùng lamivudine
4. Keát quaû veà vieäc thöïc hieän 2 quy trình ñaõ ñöôïc caûi tieán treân huyeát thanh
beänh nhaân
4.1. Xaùc ñònh ñoät bieán Basal core promoter treân huyeát thanh beänh nhaân
4.2. Xaùc ñònh ñoät bieán khaùng lamivudine treân huyeát thanh beänh nhaân
37
40
Keát luaän – Ñeà nghò
47
Taøi lieäu tham khaûo
Phuï luïc
15
22
23
25
26
33
34
35
TAØI LIEÄU THAM KHAÛO
Taøi lieäu Tieáng Vieät:
1. Nguyeãn Höõu Chí, Beänh vieâm gan sieâu vi, trang 69-140, NXB ITAXA 1993.
2. Hoà Huyønh Thuøy Döông, Sinh hoïc phaân töû, NXB Giaùo duïc, 1997.
3. Hoà Huyønh Thuøy Döông, Kyõ thuaät Di truyeàn, Giaùo trình sau ñaïi hoïc 2005.
4. Ñinh Daï Lyù Höông, Buøi Höõu Hoaøng, Traàn Thieän Tuaán Huy, Traàn Ngoïc Baûo,
Vieâm gan sieâu vi B – töø caáu truùc sieâu vi ñeán ñieàu trò, NXB Ñaø Naüng, 2000.
5. Tröông Thò Xuaân Lieân, Virus hoïc, Baøi giaûng sau ñaïi hoïc 2005.
6. Phaïm Hoaøng Phieät, Dieãn bieán töï nhieân cuûa nhieãm sieâu vi vieâm gan B maïn tính,
Sinh hoaït thöôøng kyø laàn II Hoäi gan maät TP HCM, 1999.
7. Tröông Baù Trung, Ñieàu trò theâm Adefovir dipivoxyl treân beänh nhaân vieâm gan B
khaùng lamivudine, Hoäi nghò Tieáp caän ña phöông vôùi beänh vieâm gan virus B/C, TP
HCM 10/2006.
8. Cao Vaên Vieân, Beänh vieän Vieät Ñöùc, Nhöõng ñieàu caàn bieát khi bò nhieãm vieâm
gan B, vietduchospital.edu.vn.
Taøi lieäu Tieáng Anh:
9. Allen MI, Two sensitive PCR-based methods for detection of Hepatitis B virus
variants associated with reduced susceptibility to Lamivudine, Journal of Clinical
microbiology, 1999, p. 3338-3347.
10. Chang TT, Lai CT, Four years of lamivudine treatment in Chinese patients
with chronic hepatitis B, J. Gastroenterol Hepatol 2004;19:1276-1282.
11. Clavel F, Hance AJ, HIV drug resistance, N Engl J Med 2004; 350:1023-1035.
12. Dan Yang H, PCR restriction fragment length polymorphism in detection of
YMDD variants of viral polymerase in hepatitis B virus patients treated with
lamivudine, 2002.
13. Dary T., Michael Craig, Outcomes after HBV treatment failure due to
resistance, Clinical Care Options 2006.
14. Doo E., Liang TJ, Molecular anatomy and pathophysiologic implications of
drug resistance in hepatitis B virus infection, Gastroenterology 2001;120:10001008.
15. Zoulim F., Hepatitis B virus resistance to antivirals, Inserm U271. 25.
Hepatology 2001; 34:1225-1241.
16. Zoulim F., Michael Craig, Tools for the assessment of HBV resistance in
clinical practice, Clinical Care Options, 3, 2006.
17. Tacke F., Hannover medical school, Basal core promoter and precore
mutations in the hepatitis B virus genome enhance replication efficacy of
lamivudine resistant mutants, 03/2004.
18. Gastroenterol 16, Washington D.C, 03/2001.
19. Geoffrey M., Michael Craig, Role of HBV DNA in patient management,
Clinical Care Options, 2, 2006.
20. George K.K, Michael Craig, Natural history of chronic HBV infection in
patients receiving anti-HBV therapy, Clinical Care Options, 6, 2006.
21. Gerald Y., Antonio Giulivi, Hepatitis B viral mutants and their relevance to the
health care system, 9, 2001.
22. Gish RG, Leung N, Safety and antiviral activity of emtricitabine (FTC) for the
treatment of chronic hepatitis B infection: a two-year study, J. Hepatol
Jul.2005;43(1): 60-68
23. Jake Liang T, Michael Craig, Summary of mutations associated with resistance
to HBV drugs, Clinical Care Options, 7, 2006.
24. Kakumu S., Prevalence of hepatitis B, hepatitis C and GB virus/ hepatitis G
virus in liver disease patients and in habitants in Ho Chi Minh City, VietNam, J
Med Virol 54, pp. 243-248.
25. Kenji Abe, Naoto Aiba, Complete nucleotide sequence of hepatitis B virus,
6/2003.
26. Kenji Abe, Tran Thien Tuan Huy, Characteristics of core promoter and precore
stop codon mutants of hepatitis B virus in Viet Nam, Journal of medical virology
2004,74:228-236.
27. Kenji Abe, Traàn Thieän Tuaán Huy, Molecular epidemiology of Hepatitis B and
C virus infection in Asia, Pediatrics International, 2004.
28. Lai CL, Dienstag J, Prevalence and clinical correlates YMDD variants during
lamivudine therapy for patients with chronic hepatitis B, Clin Infect Dis
2003;36:687-696.
29. Lai CL, Leung N, A 1-year trial of telbivudine, lamivudine, and the
combination in patients with Hepatitis B e antigen-positive chronic Hepatitis B,
Gastroenterology 2005;129:528-536.
30. Levine, 2002, Efficacies of entecavir against lamivudine-resistant Hepatitis B
virus replication and recombinant polymerases in vitro, Antimicrobial agents and
chemotherapy, Aug.2002, p.2525-2532.
31. Liaw YF, Chien RN, Acute exacerbation and hepatitis B virus clearance after
emergence of YMDD motif mutation during lamivudine therapy, Hepatology
1999,30:567-572.
32. Locarnini S, Molecular virology and the development of resistant mutants:
implications for therapy, Semin Liver Dis. 2005(suppl 1):9-19.
33. Locarnini S, Michael Craig, Assessing HBV virologic markers, Clinical Care
Options 2006.
34. Lok AS, Lai CL, Long-term safety of lamivudine treatment in patients with
chronic hepatitis B, Gastroenterology 2003; 125:1714-1722.
35. Maria H., Hepatitis B treatment – new directions for future therapy.
36. Melegan M., hepatitis B virus mutants associated with 3TC and famciclovir
administration are replication defective, Hepathology 27, pp628-633.
37. Meyer PR, Matsuura SE, Differential removal of thymidine nucleotide
analogues from blocked DNA chains by human immunodeficiency virus reverse
transcriptase in the presence of physiological concentrations of 2’-deoxynucleoside
triphosphates, Antimicrob Agents Chemother 2000; 44:3465-3472.
38. Naeger LK, Margot NA, Increased drug susceptibility of HIV-1 reverse
transcriptase mutants: analysis of enzyme processivity, chain-terminator removal
and viral replication, Antivirther 2001; 6:115-126.
39. Paul Martin, Michael Craig, Natural history of hepatitis B virus infection,
Clinical Care Options 2006.
40. Perrillo R., Hepatitis B – Treatment strategies for current available drugs,
Current hepatitis reports, Volume 2, Number 2, 05/2003.
41. Qi X, Ray AS, In vitro characterization of anti-HBV afficacy and intracellular
metabolism of tenofovir, J. Hepatol 2005; 42(suppl 2):A75.
42. Robinson W.S., Hepatitis B virus and hepatitis D virus – principles and practice
of infectious diseases, 1995, 1406-1428.
43. Schuurman R, Nijhuis M, Rapid changes in human immunodeficiency virus
type 1 RNA load and appearance of drug resistance virus populations in persons
treated with lamivudine (3TC), J Infect Dis 1995;171:1411-1419
44. Stephen Locarnini, Molecular virology and the development of resistant
mutants: implications for therapy, Liver disease 25, 2005.
45. Steven Han, UCLA Division of Digestive Diseases, Liver 411 Educational
articles, 2003.
46. Sugauchi F., Epidemiologic and virologic characteristic of hepatitis B virus
genotype B having the recombination with genotype C, Gastroenterology 2003,
124:925-932.
47. Sugauchi F., Hepatitis B virus of genotype B with or without recombination
with genotype C over the precore region plus the core gene, J Virol 2002, 76:59855992.
48. Tanaka T., Okamoto H., Hepatitis B virus strains with mutations in the core
promoter, Hepatology 2001; 33:1527-1532.
49. Takahashi K., The precore/core mutant T1762A1764 of hepatitis B virus: clinical
significance and an easy method for detection, 1995, J Hepatol. 2005, 43:60-66.
50. Tenny DJ., Levine SM., Clinical emergence of entercavir resistant hepatitis B
virus requires additional substitutions in virus already resistant to lamivudine,
Antimicrob Agents Chemother 2004;48:3498-3507.
51. Yuen MF, Sablon E, Factors associated with hepatitis B virus DNA
breakthrough in patients receving prolonged lamivudine therapy, Hepatology
2001;34:785-791.
52. Zarski 2004, Hepatitis B: Treatment strategies for current available drugs.
Josee Gauthier – Eric J. Bourne, Quantitation of hepatitis B viremia and
emergence of YMDD variants in patients with chronic hepatitis B treated with
lamivudine, The journal of infectious disease 1999, 180:1757-62.
53. World Health Organization Fact Sheet/204, Hepatitis B, 2000.
53. Kenji Abe, Characteristic of HBV in Ghana: Full length genome sequences
indicate the endemecity of Genotype E in West Africa, Journal of Medical
Virology 78, 2006, 178-184.
PHUÏ LUÏC:
Phuï luïc 1: Keát quaû ñònh löôïng HBV DNA so saùnh
Type
Identifier
Rep
Ct
Log
SQ
SQ
Mean
6.000
1.00E+06
1.00E+06
N/A
3
26.05
4.000
1.00E+04
1.00E+04
N/A
4
33.14
2.000
1.00E+02
1.00E+02
N/A
1
29.28
3.109
1.29E+03
1.29E+03
N/A
2
28.41
3.365
2.32E+03
2.32E+03
N/A
3
26.88
3.820
6.60E+03
6.60E+03
N/A
4
23.85
4.714
5.18E+04
5.18E+04
N/A
5
23.88
4.704
5.06E+04
5.06E+04
N/A
6
23.54
4.811
5.24E+04
5.24E+04
N/A
7
21.61
5.375
2.37E+05
2.37E+05
N/A
8
22.95
4.980
9.54E+04
9.54E+04
N/A
N/A
D09 Unknown 8T
22.95
19.59
N/A
D08 Unknown 7T
21.61
2
N/A
D07 Unknown 6T
23.54
N/A
N/A
D06 Unknown 5T
23.88
1.00E+08
N/A
D05 Unknown 4T
23.85
1.00E+08
N/A
D04 Unknown 3T
26.88
8.000
N/A
D03 Unknown 2T
28.41
12.74
N/A
D02 Unknown 1T
29.28
1
N/A
C05 Standard
33.14
SD
N/A
C04 Standard
26.05
Mean
N/A
C03 Standard
19.59
SQ
SD
C02 Standard
12.74
SQ
N/A
Ct
Ct
D10 Unknown 9T
14.98
1.64E+07
N/A
11
29.33
3.093
1.04E+03
1.04E+03
N/A
12
29.14
3.151
1.42E+03
1.42E+03
N/A
13
28.65
3.295
1.97E+03
1.97E+03
N/A
14
26.08
4.055
1.13E+04
1.13E+04
N/A
15
26.80
3.841
6.94E+03
6.94E+03
N/A
16
23.60
4.699
4.49E+04
4.49E+04
N/A
17
21.76
5.331
2.14E+05
2.14E+05
N/A
18
24.62
4.487
3.07E+04
3.07E+04
N/A
19
15.09
7.326
2.01E+07
2.01E+07
N/A
20
15.54
7.171
1.48E+07
1.48E+07 N/A
N/A
E10 Unknown 9B
15.01
1.64E+07
N/A
E09 Unknown 8B
24.62
7.214
N/A
E08 Unknown 7B
21.76
15.39
N/A
E07 Unknown 6B
23.60
10
N/A
E06 Unknown 5B
26.80
N/A
N/A
E05 Unknown 4B
26.08
2.26E+07
N/A
E04 Unknown 3B
28.65
2.26E+07
N/A
E03 Unknown 2B
29.14
7.335
N/A
E02 Unknown 1B
29.33
14.98
N/A
D11 Unknown 10T
15.39
9
N/A
E11Unknown 10B
Maãu 1T ñeán 10T: Maãu theo thöù töï 1 ñeán 10, taùch chieát baèng phöông phaùp Tuûa
Maãu 1B ñeán 10B: Maãu theo thöù töï 1 ñeán 10, taùch chieát baèng phöông phaùp Boom
Phuï luïc 2: Ñoái chieáu keát quaû giaûi trình töï vaø keát quaû PCR-RFLP phaân tích ñoät
bieán Basal core promoter
Kí
hieäu
Trình töï gen hoang daïi
(1758-TTAAAGGTCT-1767)
Keát quaû
Keát quaû
giaûi trình
PCR-RFLP
Keát luaän
maãu
töï gen
BCP01
-TTAAAGGTCT-
Hoang daïi
Hoang daïi
Phuø hôïp
BCP02
-TTAATGATCT-
T1762A1764
T1762A1764
Phuø hôïp
BCP03
-TTAAAGGTCT-
Hoang daïi
Hoang daïi
Phuø hôïp
BCP04
-TTAAAGGTCT-
Hoang daïi
Hoang daïi
Phuø hôïp
BCP05
-TTAAAGGTCT-
Hoang daïi
Hoang daïi
Phuø hôïp
BCP06
-TTAAAGGTCT-
Hoang daïi
Hoang daïi
Phuø hôïp
BCP07
-TTAAAGGTCT-
Hoang daïi
Hoang daïi
Phuø hôïp
BCP08
-TTAAAGGTCT-
Hoang daïi
Hoang daïi
Phuø hôïp
BCP09
-TTAAAGGTCT-
Hoang daïi
Hoang daïi
Phuø hôïp
BCP10
-TTAAAGGTCT-
Hoang daïi
Hoang daïi
Phuø hôïp
BCP11
-TTAAAGGTCT-
Hoang daïi
Hoang daïi
Phuø hôïp
BCP12
-TTAATGATCT-
T1762A1764
T1762A1764
Phuø hôïp
BCP13
-TTAATGATCT-
T1762A1764
T1762A1764
Phuø hôïp
Phuï luïc 3: Moät soá keát quaû ñoái chieáu giöõa giaûi trình töï vaø PCR-RFLP
phaân tích taïi codon rt180
Kí
Trình töï gen hoang daïi
Keát quaû
Keát quaû
PCR-RFLP
Keát luaän
hieäu
CAGTCCGTTTCTCCTTGGCT
giaûi trình
maãu
(Pol nu seq 657-----------676)
töï gen
KL01
CAGTCCGTTTCTCCATGGCT
180 LÆM
180 LÆM
Phuø hôïp
KL02
CAGTCCGTTTCTCCTTGGCT
180wt
180wt
Phuø hôïp
KL04
CAGTCCGTTTCTCCTTGGCT
180wt
180wt
Phuø hôïp
KL05
CAGTCCGTTTCTCCTTGGCT
180wt
180wt
Phuø hôïp
KL06
CAGTCCGTTTCTCCTTGGCT
180wt
180wt
Phuø hôïp
KL07
CAGTCCGTTTCTCCTTGGCT
180 LÆM
180 LÆM
Phuø hôïp
KL08
CAGTCCGTTTCTCCATGGCT
180wt
180wt
Phuø hôïp
KL09
CAGTCCGTTTCTCCATGGCT
180 LÆM
180 LÆM
Phuø hôïp
KL12
CAGTCCGTTTCTCCATGGCT
180 LÆM
180 LÆM
Phuø hôïp
KL13
CAGTCCGTTTCTCCATGGCT
180 LÆM
180 LÆM
Phuø hôïp
KL15
CAGTCCGTTTCTCCTTGGCT
180wt
180wt
Phuø hôïp
KL16
CAGTCCGTTTCTCCATGGCT
180 LÆM
180 LÆM
Phuø hôïp
KL17
CAGTCCGTTTCTCCTTGGCT
180wt
180wt
Phuø hôïp
Phuï luïc 4: Moät soá keát quaû ñoái chieáu giöõa giaûi trình töï vaø PCR-RFLP
phaân tích taïi codon rt204
Kí
Trình töï gen hoang daïi
Keát quaû
Keát quaû
Keát luaän
hieäu
GGCTTTCAGTCATATGGATGAT
giaûi trình
PCR-
maãu
(Pol nu seq 729--------------750)
töï genï
RFLP
KL01
GGCTTTCAGTCATGTGGATGAT
204 MÆV
204 MÆV
Phuø hôïp
KL02
GGCTTTCAGTCATATTGATGAT
204 MÆI
204 MÆI
Phuø hôïp
KL04
GGCTTTCAGTCATATGGATGAT
204wt
204wt
Phuø hôïp
204 MÆV
(thieáu V)
204wt
Phuø hôïp
204 MÆV
(thieáu V)
(thieáu daïng ñoät bieán 204 MÆV)
KL05
GGCTTTCAGTCATATGGATGAT
(thieáu daïng ñoät bieán 204 MÆV)
204wt
KL06
GGCTTTCAGTCATATTGATGAT
204 MÆI
204 MÆI
Phuø hôïp
KL07
GGCTTTCAGTCATGTGGATGAT
204 MÆV
204 MÆV
Phuø hôïp
KL08
GGCTTTCAGTCATATTGATGAT
204 MÆV
204 MÆI
Phuø hôïp
KL09
GGCTTTCAGTCATGTGGATGAT
204 MÆV
204 MÆV
Phuø hôïp
KL12
GGCTTTCAGTCATATTGATGAT
204 MÆI
204 MÆI
Phuø hôïp
KL13
GGCTTTCAGTCATGTGGATGAT
204 MÆV
204 MÆV
Phuø hôïp
KL15
GGCTTTCAGTCATATTGATGAT
204 MÆV
204 MÆI
Phuø hôïp
KL16
GGCTTTCAGTCATGTGGATGAT
204 MÆV
204 MÆV
Phuø hôïp
KL17
GGCTTTCAGTCATATGGATGAT
204wt
204wt
Phuø hôïp
Phuï luïc 5: Moät trình töï boä gen cuûa HBV duøng ñeå so saùnh
AUTHORS Takahashi,K., Brotman,B., Usuda,S., Mishiro,S. and Prince,A.M.
TITLE Full-genome sequence analyses of hepatitis B virus (HBV) strains
recovered from chimpanzees infected in the wild: implications for
an origin of HBV
JOURNAL Virology 267 (1), 58-64 (2000)
MEDLINE 20115968
REFERENCE 2 (bases 1 to 3182)
AUTHORS Mishiro,S.
TITLE Direct Submission
JOURNAL Submitted (16-SEP-1999) Shunji Mishiro, Toshiba General Hospital,
Department of Medical Sciences; 6-3-22 Higashi Oh-i, Shinagawa,
Tokyo 140-8522, Japan (E-mail:
[email protected])
ORIGIN
1 aactccacaa cattccacca agctctgcta gaccccagag taaggggcct atactttcct
61 gctggtggct ccagttccgg aacagtaaac cctgttccga ctactgcctc acccatatcg
121 tcaatcttct cgaggactgg ggaccctgca ccgaacatgg agaacacaac atcaggattc
181 ctaggacccc tgctcgtgtt acaggcgggg tttttcttgt tgacaagaat cctcacaata
241 ccacagagtc tagactcgtg gtggacttct ctcaattttc tagggggagc acccacgtgt
301 cctggccaaa attcgcagtc cccaacctcc aatcactcac caacctcttg tcctccaatt
361 tgtcctggct atcgctggat gtgtctgcgg cgttttatca tattcctctt catcctgctg
421 ctatgcctca tcttcttgtt ggttcttctg gactaccaag gtatgttgcc cgtttgtcct
481 ctacttccag gaacatcaac taccagcacg ggaccatgca agacctgcac gattcctgct
541 caaggcacct ctatgtttcc ctcttgttgc tgtacaaaac cttcggacgg aaactgcact
601 tgtattccca tcccatcatc ctgggctttc gcaagattcc tatgggagtg ggcctcagtc
661 cgtttctcct ggctcagttt actagtgcca tttgttcagt ggttcgtagg gctttccccc
721 actgtttggc tttcagttat atggatgatg tggtattggg ggccaagtct gtacaacatc
781 ttgaatccct ttttacctct attaccaatt ttcttttgtc tttgggtata catttgaacc
841 ctaataaaac caaacgttgg ggctactccc ttaacttcat gggatatgta attggaagtt
901 ggggtacttt accgcaagac catattatac taaaactcaa gcaatgtttt cgaaaactgc
961 ctgtaaatag acctattgat tggaaagtat gtcagagaat tgtgggtctt ttgggctttg
1021 ctgccccttt tacacaatgt ggctatcctg ccttaatgcc tttatatgca tgcatacaat
1081 ctaagcaggc tttcactttc tcgccaactt acaaggcctt tctgtgtaaa caatatctga
1141 acctttaccc cgttgcccgg caacggtcag gtctctgcca agtgtttgct gacgcaaccc
1201 ccactggatg gggcttggct attggccatc gccgcatgcg tggaaccttt gtggctcctc
1261 tgccgatcca tactgcggaa ctcctagcag cttgttttgc tcgcagccgg tctggagcga
1321 aactgatcgg aacagacaac tctgttgttc tctctcggaa atacacctcc tttccatggc
1381 tgctagggtg tgctgccaac tggatcctgc gcgggacgtc ctttgtttac gtcccgtcgg
1441 cgctgaatcc cgcggacgac ccgtctcggg gccgtttggg tctctaccgt ccccttcttc
1501 atctgccgtt ccggccgacc acggggcgca cctctcttta cgcggtctcc ccgtctgtgc
1561 cttctcatct gccggtccgt gtgcacttcg cttcacctct gcacgtcgca tggagaccac
1621 cgtgaacgcc caccaggtct tgcccaaggt cttacataag aggactcttg gactctcagc
1681 aatgtcaacg accgaccttg aggcatactt caaagactgt ttgtttaagg actgggagga
1741 gttgggggag gagattaggt taaaggtctt tgtactagga ggctgtaggc ataaattggt
1801 ctgttcacca gcaccatgca actttttcac ctctgcctaa tcatctcatg ttcatgtcct
1861 actgttcaag cctccaagct gtgccttggg tggctttggg gcatggacat tgacccgtat
1921 aaagaatttg gagcttctgt ggagttactc tcttttttgc cttctgactt ctttccttct
1981 attcgagatc tcctcgacac cgcttctgct ctgtatcggg aggccttaga gtctccggaa
2041 cattgttcac ctcaccatac agcactcagg caagctattc tgtgttgggg tgagttgatg
2101 aatctggcca cctgggtggg aagtaatttg gaagacccag catccaggga attagtagtc
2161 agctatgtca atgttaatat gggcctaaaa atcagacaac tactgtggtt tcacatttcc
2221 tgtcttactt ttggaagaga aactgttctt gagtatttgg tgtcttttgg agtgtggatt
2281 cgcactcctc ctgcttacag accaccaaat gcccctatct tatcaacact tccggaaact
2341 actgttgtta gacgacgagg caggtcccct agaagaagaa ctccctcgcc tcgcagacga
2401 aggtctcaat cgccgcgtcg cagaagatct caatctcggg aatctcaatg ttagtatccc
2461 ctggactcat aaggtgggaa actttactgg gctttattct tctactgtac ctgtctttaa
2521 tcctgagtgg caaactccct cctttcctca cattcattta caggaggaca ttattaatag
2581 atgtcaacaa tatgtgggcc ctcttacagt taatgaaaaa aggagattaa aattaattat
2641 gcctgctagg ttctatccta accttaccaa atatttgccc ttggacaaag gcattaaacc
2701 atattatcct gaacatgcag ttaatcatta cttcaaaact aggcattatt tacatactct
.PDF 
73 
130 
60 
