O'IZZ-I ! 2010 ^ BOYTli BAO CAO SAK PHAM NGIHtN ClJlJ »i TM CiP Bd T«nd^t^i: NGHlfiN CCti tJMG DUNG Qtl TRINH sAiN XUIT KIt4NG HUYIT TH4NH sen, B^ LifiT, ROTA SlJ DIING KJfiM B.INH vic XIN Citii nhl^ S4 Ui: PGS. TS. U TW Luto Cor quan tj6 chite) cM tri dS tM: TruBg tdm NgWto «hi Sia xaa'f Vfc xin vh Sinh ph&i Y t«f Mas6Mm{aine&y. Niton-2010 BOYTE —BoCaca— BAO CAO SAN PHAM NGHIEN CUtJ DE TAI CAP BO TSn de tai: NGHIEN ciru ifNG DVNG QUI TRINH SAN XUAT KHANG HUYET THANH Sdl, BAI LIET, ROTA SLT DVNG KIEM DINH VAC XIN Chu nhiem de tai: PGS.TS.LI Thi LuSn Off quan (Th cbvc) chii tri dl tai: Trung tam Nghien ciru San xuat Vac xin va Sinh pham Y te Ma so dl tki (neu co): Nam 2010 BOYTE sofi3ca BAO CAO SAN PHAM NGHIEN ciTu DE TAI CAP BO Ten de tai: NGHIEN CCtJ ITNG DVNG QUI TRINH SAN XUAT KHANG HUYET THANH sen, B^I LIIPT, ROTA SU^ DyNG KIEM DINH VAC XIN Chii nhifm de tai: Co* quan chu tri de tai: Cap quan ly: Ma s^ dk tki (n^u co): Then gian thu-c hi^n: Tong kinh phi thifc hi^n de tai: Trong d6: kinh phi SNKH : Ngucin kh^c (neu c6): PGS.TS.Le Thj Luan Trung tSm Nghien cihi San xuat Vac xin va Sinh pham Y te BOYte tir thang 7 nam 2007 den thang 12 nam 2009 910 trifu dong 598 tri^u dong tri^u dong Nam 2010 Bao cao sdn pham nghien ciru de tai cap Bg 1 T£n de tai: NGHIEN cut) iTNG DyNG QUI TRINH SAN XUAT KHANG HUYET THANH SOI, BAI LIET, ROTA SU^ DUNG KIEM DINH VAC XIN 2. Chu nhifm dl tai: PGS.TS.Le Thi Luan 3. Co quan chu tri de tai: Trung tam Nghien cuu San xudt Vic xin va Sinh phAm Y t^ 4. Cff quan quan ly de tai: Bg Y t^ 5. Thir ky detail 6. Pho chu nhifm de tai ho|lc ban chii nhifm de tai (neu co): 7. Danh sach nhirng ngiroi thirc hifn chinh: Nguyen Dang Hien Nguyen Thi Quy Le Trung Dung Nguyen Thiiy Huang Nguyen Thi Mai Huong Tran Bich Hanh Dang Mai Dung Nguyen Nu Anh Thu Ngo Thu Huang 8. C^c de t&i nhanh (de muc) ciia de tai (neu co) 9. Thoi gian thyc hien de tai: tu thang 7 nam 2007 d6n thang 12 nam 2009 Danh muc san pham 1. Qui trinh cong nghf san xuat huyet thanh soi 2. Qui trinh cong nghe san xuat huyet thanh bai lift 3. Qui trinh cong nghf san xuat huyet thanh Rota 4. Tieu chuan ca stf huyet thanh soi 5. Tieu chuan ca sa huyet thanh b^i lift 6. Tieu chuan ca so* huyet thanh Rota 7. Bai bao da dang 8. Dao t^o Thac sy I. Qui trinh san xuat huyet thanh sdi Nguyen vft lieu Khang nguyen Soi tit chung Edmonston do vien Kitasato cung cap; Tho trong lugng ttr 1,8 ~ 2,5 kg; Te bao Veto do vien Kitasato-Nhat ban cung cip; Moi trucmg hoa ch4t: MEM, NaHCOs 7,5%, BS, PBS (-), khang sinh P-S, Hank, chit bao quan vacxin, ta dugc tiem dap ling mien dich (Adjuvant): Ta dugc hoan chinh (CFA) va ta dugc khong hoan chinh(IFA) Vat lieu : Bom kim tiem trgn ta dugc, tu im CO2, pipet Aid, pipetman, diu tip cac loai, may ly tam lanh, ly tam thucmg, chai chiia mau (100 ml, 500 ml ), phiSn 24 giSng. Dung cu lay mau tho, Phuong pkdp Sdn xuai khdng nguyen Sdi Virus Edmonston Vcro7 da giam dgc tren 1 s6 loai li bao Vien Kitasato, mgt trong so do la te bao Vero 7 lan tnigc khi chuyen sang Viet Nam, virut nay ti6p tyc dugc ciy truy6n 2 lin lien tiip tren tl bao Vero theo qui trinh san xuit khang nguyen cua vien Kitasato Nhtit ban ggi la chung Edmonston Vero 7/P2. Hdm luang khdng nguyen: 10''°CClD50/0,5ml Tieu chuan tho sir dung: Khang the soi truac khi thii nghiem <4 don vi; Khoe manh, khong bj bat ky benh ngoai da nao; Can nang: 2,0kg den 2,5 kg; Tho dugc each ly tu 7-10 ngay Qui trinh gay ddp irng mien dich tren tho Phucmg phap vien Kitasato-Nhat ban (Khang nguyen khong tinh che) Stt 1 2 3 4 Ngay 0 07 14 28 Ngay thuc hien 10-09-2008 17-09-2008 24-09-2008 08-10-2008 Lay mau5 ml (Kiem tra khang the) Lay mau Mo Lay mau Ml (Lay mau giira ky) Cach tiem Tiem(l ) 2,5 + 2,5 (ml) (VRS+tadugc FCA) Tiem trong da nhieu vi tri, 0,1 ml/1 vi tri Tiem (2) 5 (ml) VRS khong co chat phu gia Tiem vao duoi da dui 2 ben, moi ben 2,5 ml Tiem (3) 5 (mi) VRS khong co chat phu gia Tiem vao dual da dilii 2 ben, moi ben 2,5 ml Tiem (4) 10 (ml) VRS khong c6 chat phu gia Tiem vao duoi da diii 2 ben, moi ben 5 ml 1. Qui trinh san xuat huyet thanh soi Nguyen vat tifu Khang nguyen Soi tir chung Edmonston do vien Kitasato cung cip; Tho trong lugng tir 1,8 ~ 2,5 kg; Te bao Vero do vien Kitasato-Nhat ban cimg cip; Moi trucmg hoa chit: MEM, NaHCOj 7,5%, BS, PBS (-), khang sinh P-S, Hank, chit bao quan vacxin, ta dugc tiem dap ling miin dich (Adjuvant): Ta dugc hoan chinh (CFA) va ta dugc khong hoan chlnh(IFA) Vat lieu : Bom kim tiem trgn ta dugc, tu im CO2, pipet Aid, pipetman, diu tip cac loai, m4y ly tam lanh, ly tam thucmg, chai chira mlu (100 ml, 500 ml ), phiin 24 gilng. Dung cu lay mau tho, Phutfttgphdp Sdn xudt khdng nguyen Sdi Virus Edmonston Vero7 da giam dgc tren 1 so loai tl bao Vien Kitasato, mot trong so do \k te bao Vero 7 lin tmoc khi chuyin sang Viet Nam, virut nay tilp tuc dugc ciy truyen 2 lan lien tilp tren tl bao Vero dieo qui trinh san xuit khang nguyen cua vien Kitasato Nhat ban ggi la chung Edmonston Vero 7/P2. Hdm lugng khdng nguyen: 10'°CClD50/0,5ml Tieu chuin tho sir dung: Khang thi soi truoc khi thu nghiem <4 don vi; Khoe manh, khong bi bit ky benh ngoai da nao; Can nang: 2,0kg din 2,5 kg; Tho dugc each ly tir 7-10 ngay Qui trinh gay ddp ting mien dich tren tho Phuong phap vien Kitasato-Nhat ban (Khang nguyen khong tinh chi) Stt 1 2 3 4 Ngay 0 07 14 28 Ngay thirc hien 10-09-2008 17-09-2008 24 - 09 - 2008 08-10-2008 Lay rnau5 ml (Kiem tia khang the) Lay mau Mo Lay mau Ml (Lay mau giira ky) Cach tiem Tiem (1 ) 2,5 + 2,5 (ml) (VRS+tadugc FCA) Tiem trong da nhieu vj tri, 0,1 ml/1 vi tri Tiem (2) 5 (ml) VRS khong co chat phu gia Tiem vao ducyi da diii 2 ben, m6i ben 2,5 ml Tiem (3) 5 (ml) VRS khong co chat phu gia Tiem vao duoi da diii 2 ben, moi ben 2,5 ml Tiem (4) 10 (ml) VRS khong co chat phu gia Tiem vao duoi da ddi 2 ben, moi ben 5 ml 5 6 42 49 22-10-2008 29 - 10 - 2008 Lay mau M2 (Lay het mau) Tiem (5) 10 (ml) VRS khong co chat phu gia Tiem vao tinh mach tai Gay mien dich miii 1, tiem 2,5 ml khang nguyen + 2,5 ml ta dugc (ta dugc hoan toan), tiem trong da vimg lung. Gay miin djch miii 2 sau tiem miii 1 la 7 ngay, tiem 5 ml khang nguyen, tiem duoi da diii. Gay miin djch mOi 3 sau tiem miii 2 la 7 ngay, tiem 5 ml khang nguyen, tiem duoi da diii. Gay mien dich mui 4 sau tiem mui 3 la 14 ngay, tiem 10 ml khang nguyen, tiem dual da diii. Gay mien dich mui 5 sau tiem mui 4 la 14 ngay, tiem 10 ml khang nguyen, tiem tinh mach tai. Phuong phap cua CDC-Hoa ky (Khang nguyen tinh sach qua duone) Stt 1 2 3 4 5 6 7 Ngay 0 14 21 28 35 49 Ngay thuc hien 10-09-2008 24 - 09 - 2008 01-10-2008 08-10-2008 15-10-2008 29-10-2008 Lay mau5 ml tinh mach tai (Kiem tra khang thi) Lay mau Mo Lay mau Ml Lay mau M3 (Lay het mau) Tiem Tiem (1 ) MV + CFA (1ml / con , tiem trong da lung) Tiem (2 ) MV + IFA (1ml / con , tiem trong da lung) Tiem (3 ) MV + IFA (1ml / con, tiem trong da lung) Liy mau kiem tra huyet thanh sgi 3 lan, lan 1 truoc khi gay mien dich mui 1; Lan 2 lay mau tai truoc khi gay miin dich miii 4 de kiem tra kha nang tao huyet thanh giita ky; Lan 3 liy mau toan bg sau 7 ngay gay mien dich mui 5 (ngay thii 49 ke tir ngay gay mien djch miii 1) Tai ngay thir 49 liy toan bg mau tho vao chai det, bao quan 4°C qua dem , tach huyet thanh, ly tam 3000 vong / phut trong 20 phiit, lay huyit thanh, sau do bao quan - SO'C cho toi khi sir dung. Xac dinh hieu gia huyit thanh bang phucmg phap trung hoa tren tl bao vero mgt lop. Huyit thanh dugc bit hoat 56°C trong 30 phut, sau do dugc pha loang bac 2 va dugc trung hoa vol 100 dcm vj khang nguyen soi, hin dich trung hoa dugc gay nhiim tren te bao vero 1 lop, sau 2 tuin dgc kit qua. Hieu gia khang thi la nlng do huyit thanh pha loang nhit ma tji d6 tjo ra phan img trung hoa (tl bao binh thutmg khong bi huy hoai) 2. Qui trinh san xuat huyet thanh bai liet Nguyen li(u va dung cu. Khang nguyen Bai liet typ I, II, III Ta dugc (Freunds complete adjuvant - FCA, Freunds incomplete adjuvant -FIA) Th6:Khang thi soi targe khi thir nghiem <4 dcm vi; Khoe manh, khong bi bit ky benh ngoai da nao;Can nSng: 2,0kg din 2,5 kg; Tho dugc each ly tir 7-10 ngay Te bao Hep2C cua to chiic Y te the giai Xi lanh diing trgn khang nguyen va ta dugc Ta dugc hoan chinh (CFA) va ta dugc khong hoan chinh(IFA) - Moi trucmg MEM 10%, 2% FBS - Trypsin Versen va mgt s6 dung djch khac May ly tam Phi&n nhua day det 96 gilng Tu im 37°C va 36°C - Pipet aid va pipetman cac loai - Hood vo trimg va cac dyng cu khac phyc vu cho nghien cim Phifffngphdp nghien cicu. Sdn xuit khdng nguyen bai lift: Nhan virut bai liet typ I, hoac typ II hoic typIII tren te bao vero mgt lop vciri nong do 1 CCIDjo trong 300 tl bao vcri moi truang gay nhiim DMEM tai nhiet dg 37°C trong 72-96 gia, gat hin djch virut khi huy hoai tl bao >90%. Tinh sifch khdng nguyen: Hon dich virut sau khi dugc dong tan 3 lan, ly tam 3000 vdng/phut trong 30 phut, lay nuac noi. Can dugc sieu am va ly tam lay nuac noi. Nuoc noi hgn lgi va dugc Igc voi co Igc lan lugt 0,65 (tm, 0,45 pm, 0,2 pm. Hon djch virut dugc sieu Igc vgi co dac 100-200 lin va dugc sieu li tiim 36.000 v6ng/phut/3 gio tai 4'^C, lay can, hoa loang can bang BMS va bao quan tai -70''C cho den khi sir dung - Cdch gdy ddp ling mien djch tgo khdng the bai li^t Khdng nguySn: virut sabin typ 1 hoac typ 2 hoac typ 3 Him liryng khdng nguyen: 10'°CCID 50/0,1ml Tho sir dyng: tieu chuin nhu tho sii dung cho san xuat huyet thanh scri Cich gay dip irng mien djch: Phucmg phap CDC-Hoa Ky Stt 1 2 3 4 5 6 7 Ngay 0 14 28 42 56 70 84 Lay mau Lay mau5 ml tinh mach tai (Kiem tra khang the) Lay mau Mo LiylnauMl Lay mau M2 Lay mau M3 Lay mau M4 Lay mau M5 Lay mau M6 (Lay het mau) Cach tiem Tiem (1) 0,2 + 0,2 (ml) (VRRT+la dugc FCA) Tiem trong da nhieu vi tri, 0,1 ml/1 vi tri Tiem (2) 1 + 1 (ml) (VRRT+ta dugc FIA) _ Tiem vao co diii 2 ben, moi ben 2 vj tri, moi vi ta' 0,5 ml Tiem (3) 1 + 1 (ml) (VRRT+ta dugc FL\) Tiem vao co diii 2 ben, mil ben 2 vi tri, mli vi tri 0.5 ml Tiem (4) 1 + 1 (ml) (VRRT+ta dugc FIA) Tiem vao ca diii 2 ben, moi ben 2 vi tri, moi vi ta' 0,5 ml Tiem (5) 1 + 1 (ml) (VRRT+ta dugc FLA) Tiem vao co diii 2 ben, mli ben 2 vi tri, moi vi ta' 0,5 ml Tiem (6) 1 + 1 (ml) (VRRT+ta dugc FIA) Tiem vao co diii 2 ben, moi ben 2 vi ta', moi vi ta' 0,5 ml —___^ Thu huyet thanh : ly tam 3000 vong/phiit.l 0 phut. Thu huyit thanh bao quan 6 -20°C. Kiem tra hi^u gid: khang huyet thanh bang phucmg phap trung hoa vi lugng tren tl bao Hep2C [...]... tir huyet thanh Tho \kn thir nhat 51 4.1.3 Kit qua san xuat khang thS tir huyet thanh Tho lan thir hai 57 4 2 Kit qua kiem tra tinh an toan vacxin bai liet trong phong thi nghiem 63 ^•2.1 Kiem tra nguyen lieu dau cho san xuat vacxin bai Het - khi san xuat 63 Ket qua kiem tra te bao sir dung cho san xuat vacxin bai Met trong bay nam tit nam 2000 - 2006 4 2.3 Kit qua kiem tra an toan vacxin ban thanh pham... nhan ngoai lai 16.000 cho thir nghiem xic dinh tac nhan ngoai lai 1.000.000 > 100.000 Dac hieu bang thu nghiem nhan dang qua ky thuat mien dich huynh quang 7 Bai bao da dang Bit bao da dang: 01 bai Nghien cim san xult huyit thanh Soi sir dung cho kiim dinh vic xin Tap Chi...3 Qui trinh san xuat huyet thanh rota Nguyen vat U^u Khang nguyen rota typ G1P8, G4P6, RRV T^ bao than tho Rherus MA 104 Moi trucmg DMEM, 199 Tho tieu chuin Ta dugc hoan chinh (CFA) va ta dugc khong hoan chinh(IFA) Trypsin Versen va mgt s6 dung dich... tai: Nghien ciiu san xult khang huyet thanh Bai liet tren tho va danh gia tinh an toan cua vac xin Bai liet trong phong thi nghiem Chuyen nhanh: Thii y Ma si 60.62.50 Ba bao ve vol diem xult sac nam 2008 B p GIAO DIIC VA DAO TAO TRUOfNG DAI HOC NONG NGHIEP HA NOI ca NGUYEN THI NGA NGHIEN Ciru SAN XUAx KHANG THE BAI LIET TREN THO VA DANH GIA TINH AN TOAN CUA VAc XIN BAI LIET TRONG PHONG THI NGHIEM LUAN... mau M5 7 Lay mau M6 — _ _ _ _ ^ (Lay het mau) Thu huyet tiianh: ly tam 3000 v6ng/phut,10 phut Thu huyet thanh bao quan a < 84 20 C cho toi khi sii dung Kiem tra hi^u gid: khang the trung hoa bang phucmg phap ELIZA 4 Tieu chuan khang huyet thanh Soi Tieu chi dinh gia Vo trimg Hi$u gia khang huydt thanh Tinh dac hieu Tieu chuan Nhat Ban Vo trimg >4.000 Tieu chuin cff sir Vo trimg >4.000 cho thu nghiem... liet do virus i 2.2 Lich sir phan bo 5 2.3 0 Viet Nam 5 2.4 Dac diem virus bai liet 5 2.5 Benli bai liet a ngiroi 13 2.6 Tieu chuan Viet Nam ve phuonng phap kiem tra tinh an toan ctia vacxin phong bai liet 16 2.7 Vacxin phong benh bai liet 18 2.8 Nghien cuu san xuat khang the bai liet 21 3 DOI TUONG, DIA DIEM, NOl DUNG VA PHUONG PHAP NGHIEN Ciru 23 3.1 Doi tii'cmg nghien cmi 23 3.2 Dia diem 23 3.3 Noi... tai 4'*C voi n6ng do ducmg 40% Bo nuac n6i, thu can va hoa can voi TNC, sau do pha loang khang nguyen thu dugc sau cho lugng virut khoang 10^-10^ ffii/ml Phuvng phip gay dap irng mien dich tao khang the rota {CDC-Hoa Ky) Stt Ngay Lay mau Lay mau5 ml tinh mach tai (Kiem tra khang the) Lay mau Mo Cach tiem Tiem (1 ) 0,2 + 0,2 (ml) (VRRT+ta dugc FCA) Tiem trone da nhiiu vi tri 0.1 ml/1 vi tri Tiem (2) 1... loai Hood vo trung va cac dung cu khac phuc vu cho nghien curu Phuffng phap Phifffngphdp sdn xudt khdng nguyen Te bao MAI 04 mgt lap hoac te bao vero mgt lap, d?p dugc su dyng cho su nhan len cua virut rota Te bao dugc rua 2 lan vcri moi truang DMEM im, sau do dugc dat tai 2il^C it nhat 3 gia Virut dugc hoat hoa vai trypsin nong do 15^1/ml tai nhiet dp 37°C trong 1 gia, sau do hap phu virut len te bao . huyet thanh soi 2. Qui trinh cong nghe san xuat huyet thanh bai lift 3. Qui trinh cong nghf san xuat huyet thanh Rota 4. Tieu chuan ca stf huyet thanh soi 5. Tieu chuan ca sa huyet thanh . KHANG HUYET THANH SOI, BAI LIET, ROTA SU^ DUNG KIEM DINH VAC XIN 2. Chu nhifm dl tai: PGS.TS.Le Thi Luan 3. Co quan chu tri de tai: Trung tam Nghien cuu San xudt Vic xin va Sinh . KHANG HUYET THANH Sdl, BAI LIET, ROTA SLT DVNG KIEM DINH VAC XIN Chu nhiem de tai: PGS.TS.LI Thi LuSn Off quan (Th cbvc) chii tri dl tai: Trung tam Nghien ciru San xuat Vac xin va Sinh