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 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 Danh sach nhung nguai thuc hien Danh muc cac bang so lieu Danh muc cac hinh Tom tat cac ket qua nghien cuu chinh ciia de tai 6.1 Ket qua khoa hoc 6.2 Ket qua phuc vu thuc te 6.3 Ket qua dao tao 6.4 Ket qua nang cao tiem luc khoa hoc 6.5 Danh gia tinh hinh thuc hien de tai nghien cuu BAO CAO T O N G HOP 7.1 DAT VAN DE 7.2 TONG QUAN TAI LIEU NGHIEN ClTU 7.2.1 Benh viem gan B 7.2.2 Trieu chung lam sang cua benh viem gan virus B 7.2.3 Hau qua cua viem gan virus B 7.2.4 Suphan bo cac khu vuc nhilm HBV tren th^ giai 7.2.5 Virus gay benh viem gan B 7.2.5.1 Dac dien hinh thai cua virus HBV 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 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 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 PHU LUC 36 8.1 Qui trinh tach chiet ADN tong so tumau huyet 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 cac cong bo lien quan den ket qua ciia de tai bai bao 41 8.5 Ban cac minh chiing huong dan khoa luan tot nghiep dai hoc 56 8.6 Ban 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 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 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 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 DANH MUC CAC HINH STT Ten hinh Trang 01 Ban ve sir phan bo cac khu vuc bi nhiem HBV a cac miic dp khac 02 Hinh dang ngoai ciia virus HBV 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 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 tinh sach Phan ling ngung ket giiia khang huyet tho voi hat latex dupe gan HBcAg 26 14 27 15 Thii ngung ket giua huyet 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 am tinh voi kit thii phat hien HBsAg vai hat latex gan HBc 29 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 cong b4ng ky thuat PCR, dupe tach dong 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 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 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 huyet ciia tho - Sii dung hat latex da gan HBcAg len be mat de ling dung chan doan benh HBV Kit ngung ket hat latex co dp nhay dac hieu, va dp chinh xac cao so sanh voi cac phuong phap thu nhanh (phat hien HBsAg) va ELISA (phat hien khang the khang HBcAg) Kit ngung ket rat nhay trucmg hpp phat hien cac benh nhan nhiem virus HBV 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 huyet 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 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 de phat hien benh HBV cac benh vien 6.3 Ket qua dao tao - Trong khuon kho de tai, 01 sinh vien da bao ve 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 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 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 tot cac noi dung nghien ciiu cung nhu dao tao theo hpp dong so 59/HD-NCKH, thang nam 2006 De tai da mo mot huong nghien ciiu moi ling dung hat latex de phat trien cac kit chan doan benh BAO CAO TONG HOP 7,1 DAT VAN DE Viem gan 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 ty nguoi nhiem HBV, hon 350 trieu nguoi mang HBV man tinh, 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 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 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 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, hai chi thi HBsAg va ADN khong phai luc nao cung ton tai huyet nen rat anh huong toi ket qua chan doan Trong 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 mot ngan hang mau sach lam giam thieu nguy co nhiem HBV truyen mau [19], De nang cao hieu qua, tiet kiem thoi gian va tung buoc chii dpng dupe kit chan doan, tii co nhiing bien phap thich hpp nham som phong ngua benh, de tai: 'Nghien cuu ky thuat gan khdng nguyen/khdng the len be mat hat latex kich thw&c nano vd ung dung 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 Hinh sa tao sir ngung ket giira khang the va hat latex dugc phii khang nguyen Trong nhirng nam qua, vai su dai ciia nhung thiet bi hien dai nhu may anh pho, may mat dp che phii cho phep phat hien dugc sir ngung ket giira khang the va hat latex dugc phii khang nguyen va ngugc lai a mot lugng rat nho Do vay, phuang phap diing hat latex phii khang nguyen hay khang the len be mat dugc img dung hieu qua va kinh te viec chan doan benh Cac chuyen de nghien cim du kien ciia de tai (ten va noi dung chinh ciia timg chuyen de): (i) Thu thap mdu vd phan lap gen: Dua vao cac ky thuat sinh hpc phan tir chiing toi tien hanh tach chiet ADN ciia virus gay benh viem gan B Thi^t ke cap moi d^c hieu de khuyech dai dugc gen ma hoa cho khang nguyen be mat ciia virus viem gan B Tir tach dong va xac dinh trinh tu nucleotit cua gen theo phuang phap ciia Sanger (ii) Thiit ki vector biiu hien pET 21(a) mang gen md hod cho protein vd: Du^ vao trinh tu nucleotit ciia gen ma hoa cho protein v6 ciia virus viem gan B thilt kl mot vector bilu hien mang gen c4u triic tren vai mong muon bieu hien dugc mot protein tai tl hgp Vector pET 21(a) dugc sir dung la vector bleu hien mang gen ciu true vi c6 T7 promotor khoe va duoi His-Tag thuan tien cho viec tinh sach protein tai to hgp (\u)Tinh sach protein tdi to hap Protein tai t6 hap dugc bilu hien dual cac dieu kien toi uu se duac tmh sach theo phuang phap sac ky luc (iv) Tien hanh gdn khdng nguyin Hn be mat hat latex vd so' bp ddnh gid hoat tinh ciia san phdm tao dugc Cac hat latex dugc gdn protein tai t6 hop bang phuang phap hap phu thu dpng San pham gan ket dugc danh gia bang phan ung giCra khang ngu>en tren be mat cac hat latex va khang the cua nhirng nguai mang virus viem gan B va a nhung ngirai da tiem phong viem gan B 10 cau true du kien bao cao kit qua cua de tai (chi tiet hoa cac chuong muc): a) M a diu: Danh gia chung vl benh viem gan B va nhu cdu chan doan benh b) Vat lieu va phuong phap nghien cim c) Ket qua va thao luan: Bao cao kit qua thu dugc, danh gia triln vpng ciia huong nghien ciru tuong lai d) Kien nghi 11 Tinh da nganh va lien nganh ciia dl tai: - De tai lien quan den nganh/chuyen nganh: + Sinh hpc phan tir + Vat lieu nano Sinh hpc - Tinh da/li6n nganh the hien nhu the nao noi dung va qua trinh triln khai ciia dl tai: + De tai thuc hien co su kit hgp giira nhilu ITnh vuc nghien ciru: Sinh hpc phan tir, vat lieu nano sinh hpc va y hpc 12 Phuang phap luan va phuong phap khoa hpc sir dung dl tai: 12.1 Phuong phdp phan lap gen - Thiet ke moi dua vao thong tin ve trinh tu cong bo tren mang Tach chiet ADN ciia virus gay benh viem gan B Khuyech dai gen bang ky thuat PCR - Tach dong va xac dinh trinh tir 12.2 Phucmg phdp bieu hien vd tinh sgch protein tdi to hop vi khuan E Coli Thiet ke vector bieu hien mang cau true gen ma hoa cho protein v6 ciia virus viem gan B Xac dinh dieu kien toi uu cho bieu hien ciia protein tai to hgp E Coli - Tinh sach protein tai to hgp bang phuang phap sac ky luc 12.3 Phan ung ngung ket mien dich Gan protein tai to hgp vao cac hat latex Kiem tra phan irng ngirng ket giua protein tai to hgp tren hat latex va khang the 13 Su dung nhung trang thiet bi nao ciia Bp mon, ciia Khoa: Vien Cong nghe Sinh hpc 14 Kha nang hgp tac quoc te - Hpp tac da/dang co (ten to chirc va van de hgp tac): + Truong DH Heidelberg, CHLB Dire 4- Vien IPK Gatersleben, CHLB Dire - Hgp tac se co (ten to chirc va van de hgp tac): 15 Cac hoat dpng nghien ciru ciia de tai: - Nghien ciru ly thuyet 21 - Dieu tra khao sat D - Bien soan tai lieu K - Viet bao cao khoa hpc S - Hoi thao khoa hpc n - Tap huan Q - Hoat dpng khac Q 16 Ket qua du kien: 16.1 Ket qua Khoa hpc - Du kien nhirng dong gop ciia dl tai: + Tao ca sa khoa hpc va thuc tiln cho viec chi tao cac loai kit chin doan benh sir dung hat latex gan khang nguyen/khang thi irng dung y hpc cua Viet Nam - So bai bao, sach, bao cao khoa hpc du kien se dugc cong bl: + 02 bai bao Dang tren cac tap chi chuyen nganh nuoc cip quoc gia (Tap chi Cong nghe Sinh hpc, tap chi y hpc img dung ) 16.2 Ket qua img dung - San pham cong nghe: + Quy trinh san xuat cac protein khang nguyen v6 ciia virus viem gan B + Quy trinh tao cac hat latex gan cac khang nguyen tai to hgp - Kha nang irng dung thuc te ciia cac ket qua: + Ket qua thu dugc lam co so cho viec xay dung nhirng de an KHCN cap cao hon va qui mo Icm han + Mo hucmg mai nghien cim tao cac kit chan doan benh virus a nguai va dpng vat 16.3 Ket qua dao tao - S6 cir nhan, thac sT, tiln sT dugc dao tao khuon kho ciia de tai: + 01 cir nhan - Doi mai/bo sung cho noi dung cac giao trinh/chuyen de + Lam CO so ly luan cho cac bai giang tai truang dai hpc 16.4 Kit qua vl tang cuong tiem luc cho don vi - Ket qua boi duang can bp: + Tao dilu kien cho dpi ngii can bp tilp can hoc hoi huong nghien cuu moi cong tac chin doan benh bing su dung ky thuat ngung ket hat latex - Dong gop cho viec tang cuong trang thiet bi: 17 T6ng kinh phi dk nghi: 20 000 000 (hai muai trieu dong) 18 NOl DUNG VA TIEN DO THUC HIEN CUA DE TAI (CAC CONG VIEC CAN TRIEN KHAI, THCil HAN THUt HIEN VA SAN P H A M DAT DUOC) TT Thoi gian thuc hien Tu thang Den thang Hoat dong nghien cuu Thu thap va viet tong quan tai lieu Xay dung de cuong nghien cim chi tiet San pham khoa hoc Thu thap day du tai lieu de Viet tong quan Hoan ban de cuong nghien cim chi tiet Chuyen de/Npi dung Hoan viec thu thap mau va phan lap gen Thiet ke cong vector Chuyen de/Npi dung bieu hien va bieu hien cong protein v6 ciia virus viem gan B Chuyen de/Npi dung 10 Hoan viec tinh sach protein tai to hgp Hoan Chuyen de/Npi dung 11 12 viec gan protein tai to hgp nen be mat ciia cac hat latex Dieu tra khao sat, thi 12 Hoan 12 Hoan 12 Hoan Hoan 11 12 Hoan Tong ket so lieu 11 12 Hoan Viet bao cao tong hgp 11 12 Hoan Hoi thao lan cuoi 11 12 Hoan Hoan thien bao cao 11 12 Hoan 11 12 Hoan 11 12 Hoan nghiem, thu thap so lieu Xir ly ket qua Viet bao cao cac chuyen dl Hoi thao giira ky Bo sung so lieu/thir nghiem/img dung Nop scin pham Nghiem thu de tai H : i ' 19 PHAN BO KINH PHI T - rofiE^i^^^- TT Kinh phi Don vi: trieu VND Xay dung de cuong chi tiet 0,5 Thu thap va viet tong quanxtaiMleu 0,5 Thu thap tu lieu (mua; thue) '•• Dich tai lieu tham khao (s6 trang x gia) Viet tong quan tu lieu •' 0,5 Dieu tra, khao sat, thi nghiem, thu thap so lieu, nghien cim Chi phi tau xe, cong tac phi Chi phi thue muon Chi phi hoat dpng chuyen mon Thue, mua sam trang thiet bi, nguyen vat lieu 12 Thue trang thiet bj Mua trang thiet bi Mua nguyen vat lieu, hoa chat 12 Viet bao cao khoa hpc, nghiem thu Viet bao cao Hoi thao Nghiem thu I Chi khac Mua van phong pham [n an, photocopy Quan ly phi 20 000 000 Tong kinh phi Tai lieu tham khao Dinh Duy Khang, Chu Hoang Ha, Pham Thuy Hong, Nguyen Thanh Thuy, Nong Van Hai, Dai Duy Ban, Molecular cloning of Hepatitis B surface Antigen gene from human liver tumor biopsies Annual Report of the InslUuie of Biotechnology 1996, 20-25 F Javier Gella, Josep Serraa and Juan Generx, Latex agglutination procedures in immunodiagnosis Chem., Vol.63, No.8, (1991), 1131-1 134 J.M Singer and C M Plotz, the LATEX fixation Test: application to the serologic diagnosis of RHEUMATOID arthritis Am J Med 2\(\956), 888-892 L.B Bangs, Uniform Latex Particles, Monograph, Seradyn Inc,Ind,ianapolis (1987) Yi-Chemg Chen, Victoria Dimonie, Mohamed S ELAasser, Theoretical aspects of developing latex particle morphology Chem., Vol 64, No 11 (1992), 1691-1696 Sukone Pradutkanchana, MSc, Janya Nakarin, PhD, The Use of Latex Agglutination for the Diagnosis of Acute Human Leptospirosis J Med Assoc Thai 88 (10) (2005), 1395-4000 Yamashiki M, Nishimura A, Kishioka H, Takase K, Kosaka Y, Rapid determination of hepatitis B surface antigen (HBsAg) by latex agglutination using an integrating sphere turbidimetric assay, J Immunol Methods\5S(2) (1993), 256-266 Ngay thang 06 nam 2006 Thii trucmg don vi Chii tri de tai V|£N TRUC3NG S.TS LeTran Binh TS Chu Hoang Ha T/LHlEUTRl*nNG TRU'ONG PHONG DAO TAO SAf •• " •-• / fj -SAI H O C vv V COflG NGH rOiU^^y, r^KH ^ ^ ^ v ^ ^ v ^ o i A MA NQI RUONG DAI HOC CONG NGHE CONG HOA XA HOI CHU NGHIA VIET NAM Doc lap - Tu - Hanh phuc So: 5~9 /HD-NCKH """"" Ha Ndi 12 thdng nam 2006 HOP DONG THUC HIEN D ^ TAI NGHIEN CUU KHOA HOC CAP DAI HOC QUOC GIA HA NOI NAM 2006 - Cdn cir Quy dinh vi' To chuc vd hoat clgngcua Dai iwc Quoc gia Ha Not ban hanh then Quyet dinh so 600rTCCB ngdy 01 thdng 10 nam 2001 cua Dai hoc Qudc gia Ha Noi: qui dinh quyen han cua Hieu truang cdc fricang Dai hoc thdnh vicn; - Cdn cu Cong van so 192/KHCN ngdy 25 thdng nam 2006 cua Gidm doc Dai lioc Quoc gia Hd Ngi ve vice giao nhiem vu vd chi tieu ke hoach khoa hoc cdng nghe vd nwi trudng nam 2006; - Cdn cu Quyet dinh so 316/QD-NCKH ngdy 31 thdng nam 2006 cua Hieu inirhi^ Truang Dgi hoc Cong nghe DHQGHN ve viec giao nhiem vu thuc hien de tdi NCKH cdp DHQG nam 2006 Tricang quan ly cho cdc chu nhiem de idi; - Cdn cir De cuong nghien cifu cua de tdi dd dugc phe duvet Zhung toi gom: Ben giao nhiem vu (goi la Ben A): Trirang Dai hoc Cong nghe - DHQG HN Dai dien la: PGS TS Nguyen Ngoc Binh Chuc vu: Pho Hieu truang Ben nhan nhiem vu (goi la Ben B) Ong (Ba): Don vi cong tac: Dien thoai: TS Chu Hoang Ha Tmng tam Cong nghe Sinh hoc Phan tir - Tmong Dai hpc Cong nghe 7562368, Email: chuhoangha@ibt.ac.vn ky hop d6ng thuc hien dd tai nghien ciru klioa hpc cap Dai hoc Quoc gia Ha Noi: Ten de tai: "Nghien cuu ky thuat gan khang nguyen/ khang the len be mat hat latex kich thuac nano va ung dung chan doan nhanh benh o ngiroi va gia sue" Mas6:QC.06.27 vcfi nhiing dieu khoan thoa thuan nhu sau: Dieu 1: Ben B chiu trach nhiem to chirc trien kliai thuc hien cac npi dung nghien ciai ciia de tai theo dung tien dp thuc hien da dang ki de cuong nghien cuu va thuc hien day du cac nhiem vu dugc Hieu truang Trudng Dai hpc Cong nghe giao, ghi quyet dinh so 316/QDNCKH 31/5/2006 Bieu 2: Ben B nop cho ben A cac san pham khoa hpc ciia dd tai truac 31/5/2007, bao gom: - Quy trinh san xua't cac protein khang nguyen v6 ciia virus viem gan B Quy trinh tao cac hat latex gin cac khang nguyen tai to hop - 01 den 02 bai bao dang tren cac tap chi chuyen nganh - Tong quan ve d6 tai kem theo file dien tu (Mot ban b^ng tieno Viet, mot ban bans tieng Anh - Highlight; m6i ban dai khoang 400 tir tren mot trans o,av kh6 A4 fom Times New Roman, ca chu 13pt, each dong don: Noi dung: torn tlt^muc ueu phuans phap va npi dung nghien cuu, ket qua dat duoc danh g.a y nghla va tac dong khoa hoc cong nghe ciia cac ket qua dat duac cung nhu ciia viec thuc hien de tai) Dieu 3: Tong kinh phi ciia d6 tai da duac phe duyet la: 20tneu dPn^ (bang chir: Hai muai trieu dong chrin) Chi phi cu the nhu du loan ciia ban du tru kinh phi (iheo m^u cua phonu TV-KT) Dieu 4: Ben B co trach nhiem sir dung kinh phi duoc cap iheo dung muc dich, diing che lai chinh hien hanh quyet loan voi phong Tai vu - Ke loan truoc 31/5/2007 va thuc hien viec nghiem thu de tai khong cham han 60 sau thai han thuc hien de tai (truoc 31/5/2007) ghi adieu Di^u 5: Ben A giu quyen so huu doi vai cac kel qua khoa hoc cua de tai Tai ca cac con" bo lien quan den npi dung khoa hpc ciia de tai phai ghi ro nguon tai ira kinh phi nghien ciru theo ma so cua de tai nhu sau: - Doi voi bai bao, bao cao khoa hpc: "Cong trinh duoc tai tra mot phan tu de tai mang ma so: QC.06.27, Dai hpc Quoc gia Ha Npi" - Doi voi luan van (khoa luan ): "Luan van (khoa luan ) duac thuc hien voi sir h6 tra mot phan ciia de tai mang ma so: QC.06.27, Dai hpc Quoc gia Ha Noi" - Doi voi bai bao, bao cao dang o tap chi', ky yeu hoi nghi quoc te (tieng Anh): "This work is (partly) supported by the research project No QC.06.27 granted by Vietnam National University, Hanoi" Dieu 6: Hai ben cam ket thuc hien dung cac dieu khoan da ghi hcrp dong Trong qua trinh thuc hien hpp dong, hai ben co trach nhiem thong bao kip thai cho nhung van de virdng mic va ciing ban bac, tich cue tim bien phap giai quye't Dieu 7: Hop dong lam ban, moi ben giu' mot ban, hai ban giri cho phong TV-KT, mpl ban luu tai phong HC-QT DAI DIEN BEN A ^ w , / HIEU T R U a N ( T ^ DAI DIEN BEN B K- Pm HIEU TRUONG PGS TS NGUYEN NGOC BINH P O S T S L E T R A N BINH 8.7 Phu luc Tong quan ve de tai Tieng Viet Nghien cihi ky thuat gan khang nguyen/khang the len be mat hat latex kich thuac micro/nano va irng dung ch^n doan nhanh benh a ngucri va gia siic Ky thuat ngimg ket hat latex lan dau tien dupe mieu ta va sir dung vao nam 1956 bai Singer va Plotz Tir den nay, ky thuat duac img dung rong rai viec phat hien sir nhiem vi khuan, virus, benh tir mien, cac hocmon va protein huyet Cac hat latex co kich thuac tir 0.05 |am den ^m duoc phii tren be mat boi cac phan tir protein khang nguyen tai to hgp Khi mau can chan doan co chua khang the dugc trpn vai cac hat latex dong nhat thi se tao su ngung ket cue bp ma mat thuong Cling co the quan sat dugc Ban chat ciia sir ngung ket la sir tiep xiic giua khang nguyen va khang the tao sir lien ket giira khang the va khang nguyen hay chinh vai cac hat latex C) Viet Nam hien nay, viec chii dpng nghien cuu phuang phap che tao va irng dung hat latex de tao cac vat lieu de xay dimg cac kit chan doan benh la mot ITnh vuc dang dugc quan tam Gen c ma hoa cho khang nguyen loi HBcAg da dugc khuyech dai cong bing phan img PCR vai cap moi dac hieu va dugc tach dong bang vector pBT Ket qua dpc trinh tu cho th^y gen ma hoa cho HBcAg dai 639 bp va trinh tu nucleotit ciia gen dugc dang ky tren ngan hang gen EMBL vai ma so la AM888201 Gen dugc gin vao vector bilu hien pET32b de bieu hien thira vi khuan E coli Sau protein tai t6 hgp da dugc tinh sach bang bp kit ProBond Nickel-Chelating Resin Khang nguyen tai to hgp HBc dugc gan len be mat hat latex bang hen ket cong hoa tri Phan irng ngung kit giira hat latex gan HBcAg tren be mat vai khang thi khang HBcAg huylt ciia tho dugc chuan hoa truac kiem tra kha nang ngung kit vai cac mau huylt cua nguai Hat latex gan \o\ 400 f.Lg HBcAg CO kha nang phat hien khang thi nhay hon gan vai HBcAg a lugng 100 ^g va 200 i^g va dugc chpn dl kilm tra cac mau huyet cua nguai Kit ngung kit hat latex co dp nhay, dac hieu, va dp chinh xac cao so sanh \o\ cac phuang phap thu nhanh (phat hien HBsAg) va ELISA (phat hien khang the khang HBcAg) Hat latex g4n khang nguyen HBc co the bao quan a 4()C khoang thang hoac han ma khong bi mdt hoat tinh Han nira, kit ngung koi co the phai hien duac benh 72 nhan nhilm virus HBV a giai doan man tinh vdi thao tac thuc hien dan gian, kinh tl va tiet kiem thai gian Tieng Anh Study on technique for production of antigen/antibody coated micro/nano latex particles and application for rapid diagnosis test of diseases in human and animal Latex agglutination test was first described and used by Singer and Plotz in 1956 Since then, latex agglutination technique has been applied for many diagnosis test of bacteria, virus infection, hormon, rheumatoid arthritis, Latex particle which has the size of about 0.05 ^im to jam had been coated with recombinant antigens When the diagnostic samples which contain antibodies were mixed with antigen coated latex beads suspension, it will result in aggregation of latex particles (clumping of the microspheres, to look like curdled milk) that can be delected by eyes The agglutination are usually based upon the very specific interaction of antigen (Ag) with antibody (Ab) Sub-micron sized polystyrene microspheres, often called "uniform latex particles" are used for the solid support, Ab or Ag can be adsorbed onto them In Viet Nam, research on techniques to produce latex particles and application of them to develop diagnotic tests are under development By using PCR technique, HBcAg encoding gene was successfully amplified using specific primers The gene was subsequently cloned into pBT vector and sequenced Full length of this gene is 639 bp and was submitted to EMBL datadases with accession number AM888201 Gene coding HBcAg was cloned into an expression vector pET32b for overexpression of recombinant protein in E coli After production in E coli, protein was purified using ProBond Nickel-Chelating Resin kit The purified recombinant HBc antigen was attached to carboxyl-modified microspheres via covalent coupling The agglutination between the recombinant HBc antigen-coated latex beads and anti-rabbit serum was evaluated before these latex beads were tested with human sera Latex beads coated with 400 ug of the recombinant HBc antigen can detect antibodies more sensitively than the 100 fag, 200 jag-coated latex beads The new diagnosis test employing HBcAg coated latex beads was sensitive, specific and accurate as compared to the standard lateral tlow test and ELISA test The svnthesized HBcAg coated latex beads are stable and 73 could be stored at 4°C for more than three months without loosing activity Moreover, the recombinant HBc antigen-coated latex beads could detect specifically anti-HBc antibodies during chronic phase of the illness The lest is simple, inexpensive, and less time-consuming in the management of a large number of patients 74 8.8 Phu luc Phieu torn tat ket qua nghien cihi cua de tai b^ng tieng Anh SUMMARY Project Title: Study on technique for production of antigen/antibody coated micro/nano latex particles and application for rapid diagnosis test of diseases in human and animal Code number: QC.06 27 Coordinator: Dr Chu Hoang Ha Implementing Institution: Center of Molecular Biotechnology - College of Technology - Viet Nam National University Ha Noi 144 Xuan Thuy - Cau Giay - Ha Noi, Tel: 7547461, Fax: 7547460 Cooperating Institution(s): - Institute of Biotechnology, Vietnam Academy of Science and Technology Hoang Quoc Viet str 18 - Cau Giay - Hanoi Duration: from 06/2006 to 06/2007 Objectives To develop a new diagnosis test base on latex agglutination method (LAT) for detection of HBcAg antibodies Main contents + Isolation of c gene encoding for HBcAg + Construction of expressional vector and expression of HBcAg in E coli + Optimization of procedure for attaching of HBcAg to latex particles Determination of the optimal HBc antigen concentration for sensitizing latex particles + Application of these HBcAg coated latex particles for detection of antibodies to HBcAg in HBV infected patient's serum Results obtained 75 By using PCR technique, HBcAg encoding gene was successfully amplified using specific primers The gene was subsequently cloned into pBT vector and sequenced Full length of this gene is 639 bp and was submitted to EMBL datadases with accession number AM888201 Gene coding HBcAg was cloned into an expression vector pET32b for overexpression of recombinant protein in E coli After production in E coli, protein was purified using ProBond Nickel-Chelating Resin kit The purified recombinant HBc antigen was attached to carboxyl-modified microspheres via covalent coupling The agglutination between the recombinant HBc antigen-coated latex beads and anti-rabbit serum was evaluated before these latex beads were tested with human sera Latex beads coated with 400 |ag of the recombinant HBc antigen can detect antibodies more sensitively than the 100 \xg, 200 jag-coated latex beads The new diagnosis test employing HBcAg coated latex beads was sensitive, specific and accurate as compared to the standard lateral fiow test and ELISA test The synthesized HBcAg coated latex beads are stable and could be stored at 4''C for more than three months without loosing activity Moreover, the recombinant HBc antigen-coated latex beads could detect specifically anfi-HBc antibodies during chronic phase of the illness The test is simple, inexpensive, and less time-consuming in the management of a large number of patients 76 8.9 Phu luc Phieu dang ky ket qua nghien cuu KHCN Ten de tai: Nghien ciru ky thuat gin khang nguyen/khang thi len be mat hat latex kich thudc nano va irng dung chan doan nhanh benh a ngu-oi va gia siic Ma s6: QC.06 27 Co quan quan ly de tai: Dai hpc Quoc gia Ha Npi Dia chi: 144, duong Xuan Thuy, C4u Giiy-Ha Noi Dien thoai: 7548664 Co quan chii tri de tai; Trung tam Cong nghe Sinh hpc Phan tir, Dai hpc Cong nghe Tong chi phi thuc chi: 20 000 000 (Hai muai trieu dong) Trong do: - Tu ngan sach Nha nuac: 20 000 000 d (Hai muoi trieu d5ng) - Nguon khac: Khong co Thai gian nghien cim: 12 thang Thai gian bit diu: 1/06/2006 Thai gian kit thuc: 31/05/2007 Ten cac can bp phoi hgp nghien ciru: - Chii tri de tai: TS Chu Hoang Ha - Nhung nguai tham gia: PGS.TS LeTrinBinh PGS.TS Dinh Duy Khang TS Le Van Scm CN Phan Trpng Hoang SV Hoang Ha So dang ky de tai Ngay So chirng nhan dang ky kel qua nghien ciru Ngay 77 Bao mat A Pho bien rong rai B Pho bien han che C Bao mat 16m tat ket qua nghien cim: - Gen c da dugc khuylch dai cong bing phan ung PCR xa dugc tach dong vector tach dong pBT Trinh tir cua gen c dugc dang ky tren ngan hang gen EMBL vai ma s6 la AM888201 San phim ma hoa cua gen c la protein HBcAg dugc bieu hien dir thira vi khuin K coU va dugc tinh sach vol ham lugng lan Day la ngu6n protein tai lo hcrp tinh sach dugc sir dung qua trinh tao kit chan doan benh HBV dua theo nguven l> ngung kit Khang nguyen tai t5 hgp HBcAg dugc gin cong len bl mat hai latex bang lien kit cong hoa trj Lugng khang nguyen tai to hgp HBcAg thich hgp nhit de gin len bl mai hat latex la 400 - iTng dung hat latex da gin khang nguyen HBcAg dk chin doan HBV voi cac mau huylt cua nguai Phuang phap ngung kit hat latex co dp nhay, dac hieu, va dp chinh xac cao so sanh vai cac phuang phap thu nhanh (phat hien HBsAg) va ELISA (phat hien khang thi khang HBcAg) Hat latex gin khang nguyen HBc co thi bao quan o 4"C khoang thang hoSc han ma khong bj mit hoat tinh Han nua, kit ngung kit c6 thi phat hien dugc benh nhan nhiem virus HBV a giai doan man tinh vai thao tac thuc hien dan gian, kinh ll va tilt kiem thoi gian Kiln nghi ve quy mo va doi tugng ap dung ket qua nghien ciru: - San pham cua de tai, bp kit ngung kit hat latex gin HBcAg co Igi thi phat hien dugc benh HBV giai doan man tinh rat c6 y gop phin giam thieu dugc ngu\ co nhiem HBV truyen mau gay Do qui trinh tien hanh chan doan theo phuong phap ngung kit ril dan gian nhanh, chinh xac nen co the ung dung chan doan tren qui mo Idn tai cac co so \ tl, dac biet la tai cdc tuyen ca ca noi chua dugc trang bi thilt bi chin doan hien dai - Quy trinh sir dung hat latex de phat trien cac bp kit chuan doan benh dugc phat triln va su dung de tai co the la tien de cho cac nghien cuu xay dung cac bp kit chan doan benh (HIV, H5N1, ) theo nguyen tac tuong tu Chuc vu Ho va ten Hoc vi Ky ten Chu nhiem de tai Chu Hoang Ha TS Thu trucmg ca quan chii tri de tai Chu tich hoi dong danh gia chinh thuc Thu truong co quan quan l\ de tai ! PGS TS AlT Dong dau 78 ... natri dodecyl sulfat sau 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... mau huyet cho ket qua am tinh vdi kit thir phat hien HBsAg, chiing toi thu dupe mot mau cho ket qua ngung ket rat ro rang cac mau lai hoan toan khong co hien tupng ngung ket xay Dieu cho thay,... 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